Endoglin polypeptides and uses thereof

ABSTRACT

In certain aspects, the present disclosure relates to the insight that a polypeptide comprising a truncated, ligand-binding portion of the extracellular domain of endoglin (ENG) polypeptide may be used to inhibit angiogenesis in vivo, particularly in mammals suffering angiogenesis-related disorders.

RELATED APPLICATIONS

This application claims the benefit of the filing date under 35 U.S.C.§119(e) to U.S. Provisional patent application Ser. No. 61/477,585,Filed Apr. 20, 2011, entitled “Endoglin Polypeptides And Uses Thereof,”the entire contents of which are incorporated herein by reference.

BACKGROUND

Angiogenesis, the process of forming new blood vessels, is critical inmany normal and abnormal physiological states. Under normalphysiological conditions, humans and animals undergo angiogenesis inspecific and restricted situations. For example, angiogenesis isnormally observed in wound healing, fetal and embryonic development andformation of the corpus luteum, endometrium and placenta.

Undesirable or inappropriately regulated angiogenesis occurs in manydisorders, in which abnormal endothelial growth may cause or participatein the pathological process. For example, angiogenesis participates inthe growth of many tumors. Deregulated angiogenesis has been implicatedin pathological processes such as rheumatoid arthritis, retinopathies,hemangiomas, and psoriasis. The diverse pathological disease states inwhich unregulated angiogenesis is present have been categorized asangiogenesis-associated diseases.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Capillary blood vessels are composed primarily ofendothelial cells and pericytes, surrounded by a basement membrane.Angiogenesis begins with the erosion of the basement membrane by enzymesreleased by endothelial cells and leukocytes. The endothelial cells,which line the lumen of blood vessels, then protrude through thebasement membrane. Angiogenic factors induce the endothelial cells tomigrate through the eroded basement membrane. The migrating cells form a“sprout” protruding from the parent blood vessel, where the endothelialcells undergo mitosis and proliferate. Endothelial sprouts merge witheach other to form capillary loops, creating the new blood vessel.

Agents that inhibit angiogenesis have proven to be effective in treatinga variety of disorders. Avastin™ (bevacizumab), a monoclonal antibodythat binds to vascular endothelial growth factor (VEGF), is used in thetreatment of a variety of cancers. Macugen™, an aptamer that binds toVEGF has proven to be effective in the treatment of neovascular (wet)age-related macular degeneration. Antagonists of the SDF/CXCR4 signalingpathway inhibit tumor neovascularization and are effective againstcancer in mouse models (Guleng et al. Cancer Res. 2005 July 1;65(13):5864-71). A variety of so-called multitargeted tyrosine kinaseinhibitors, including vandetanib, sunitinib, axitinib, sorafenib,vatalanib, and pazopanib are used as anti-angiogenic agents in thetreatment of various tumor types. Thalidomide and related compounds(including pomalidomide and lenalidomide) have shown beneficial effectsin the treatment of cancer, and although the molecular mechanism ofaction is not clear, the inhibition of angiogenesis appears to be animportant component of the anti-tumor effect (see, e.g., Dredge et al.Microvasc Res. 2005 January; 69(1-2):56-63). Although manyanti-angiogenic agents have an effect on angiogenesis regardless of thetissue that is affected, other angiogenic agents may tend to have atissue-selective effect.

It is desirable to have additional compositions and methods forinhibiting angiogenesis. These include methods and compositions whichcan inhibit the unwanted growth of blood vessels, either generally or incertain tissues and/or disease states.

SUMMARY

In part, the present disclosure provides endoglin (ENG) polypeptides andthe use of such endoglin polypeptides as selective antagonists for BMP9and/or BMP10. As described herein, polypeptides comprising part or allof the endoglin extracellular domain (ECD) bind to BMP9 and BMP10 whilenot exhibiting substantial binding to other members of the TGF-betasuperfamily. This disclosure demonstrates that polypeptides comprisingpart or all of the endoglin ECD are effective antagonists of BMP9 andBMP10 signaling and act to inhibit angiogenesis and tumor growth invivo. Thus, in certain aspects, the disclosure provides endoglinpolypeptides as antagonists of BMP9 and/or BMP10 for use in inhibitingangiogenesis as well as other disorders associated with BMP9 or BMP10described herein.

In certain aspects, the disclosure provides polypeptides comprising atruncated extracellular domain of endoglin for use in inhibitingangiogenesis and treating other BMP9 or BMP10-associated disorders.While not wishing to be bound to any particular mechanism of action, itis expected that such polypeptides act by binding to BMP9 and/or BMP10and inhibiting the ability of these ligands to form signaling complexeswith receptors such as ALK1, ALK2, ActRIIA, ActRIIB and BMPRII. Incertain embodiments, an endoglin polypeptide comprises, consists of, orconsists essentially of, an amino acid sequence that is at least 70%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence ofamino acids 42-333, 26-346, 26-359 or 26-378 of the human endoglinsequence of SEQ ID NO:1. An endoglin polypeptide may comprise, consistof, or consist essentially of an amino acid sequence that is at least70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequenceof amino acids beginning at any of positions 26-42 of SEQ ID NO:1 andending at any of positions 333-378 of the human endoglin sequence of SEQID NO:1. An endoglin polypeptide may comprise, consist of, or consistessentially of, a polypeptide encoded by a nucleic acid that hybridizesunder less stringent, stringent or highly stringent conditions to acomplement of a nucleotide sequence selected from a group consisting of:nucleotides 537-1412 of SEQ ID NO: 2, nucleotides 121-1035 of SEQ ID NO:30, nucleotides 121-1074 of SEQ ID NO: 26, nucleotides 121-1131 of SEQID NO: 24, nucleotides 73-1035 of SEQ ID NO: 30, nucleotides 73-1074 ofSEQ ID NO: 26, and nucleotides 73-1131 of SEQ ID NO: 24. In each of theforegoing, an endoglin polypeptide may be selected such that it does notinclude a full-length endoglin ECD (e.g., the endoglin polypeptide maybe chosen so as to not include the sequence of amino acids 379-430 ofSEQ ID NO:1, or a portion thereof or any additional portion of a uniquesequence of SEQ ID NO:1). An endoglin polypeptide may be used as amonomeric protein or in a dimerized form. An endoglin polypeptide mayalso be fused to a second polypeptide portion to provide improvedproperties, such as an increased half-life or greater ease of productionor purification. A fusion may be direct or a linker may be insertedbetween the endoglin polypeptide and any other portion. A linker may bea structured or unstructured and may consist of 1, 2, 3, 4, 5, 10, 15,20, 30, 50 or more amino acids, optionally relatively free of secondarystructure. A linker may be rich in glycine and proline residues and may,for example, contain a sequence of threonine/serine and glycines (e.g.,TGGG (SEQ ID NO: 31)) or simply one or more glycine residues (e.g., GGG(SEQ ID NO: 32). Fusions to an Fc portion of an immunoglobulin orlinkage to a polyoxyethylene moiety (e.g., polyethylene glycol) may beparticularly useful to increase the serum half-life of the endoglinpolypeptide in systemic administration (e.g., intravenous, intraarterialand intra-peritoneal administration). In certain embodiments, anendoglin-Fc fusion protein comprises a polypeptide comprising,consisting of, or consisting essentially of, an amino acid sequence thatis at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toa sequence of amino acids starting at any of positions 26-42 of SEQ IDNO:1 and ending at any of positions 333-378 of the human endoglinsequence of SEQ ID NO:1, and optionally may not include a full-lengthendoglin ECD (e.g., the endoglin polypeptide may be chosen so as to notinclude the sequence of amino acids 379-430 of SEQ ID NO:1, or a portionthereof, or so as not to include any 5, 10, 20, 30, 40, 50, 52, 60, 70,100, 150 or 200 or more other amino acids of any part of endoglin or anypart of amino acids 379 to 581 of SEQ ID NO:1), which polypeptide isfused, either with or without an intervening linker, to an Fc portion ofan immunoglobulin. An endoglin polypeptide, including an endoglin-Fcfusion protein, may bind to BMP9 and/or BMP10 with a K_(D) of less than10⁻⁸M, 10⁻⁹M, 10¹⁰M, 10⁻¹¹M or less, or a dissociation constant (k_(d))of less than 10⁻³s⁻¹, 3×10⁻³s⁻¹, 5×10⁻³s⁻¹ or 1×10⁻⁴s⁻¹. The endoglinpolypeptide may be selected to have a K_(D) for BMP9 that is less thanthe K_(D) for BMP10, optionally less by 5-fold, 10-fold, 20-fold,30-fold, 40-fold or more. The endoglin polypeptide may have little or nosubstantial affinity for any or all of TGF-β1, -β2 or -β3, and may havea K_(D) for any or all of TGF-β1, -β2 or -β3 of greater than 10⁻⁹M,10⁻⁸M, 10⁻⁷M or 10⁻⁶M.

An Fc portion may be selected so as to be appropriate to the organism.Optionally, the Fc portion is an Fc portion of a human IgG1. Optionally,the endoglin-Fc fusion protein comprises the amino acid sequence of anyof SEQ ID NOs: 33, 34, 35, or 36. Optionally, the endoglin-Fc fusionprotein is the protein produced by expression of a nucleic acid of anyof SEQ ID Nos: 17, 20, 22, 24, 26, 28 or 30 in a mammalian cell line,particularly a Chinese Hamster Ovary (CHO) cell line. An endoglinpolypeptide may be formulated as a pharmaceutical preparation that issubstantially pyrogen free. The pharmaceutical preparation may beprepared for systemic delivery (e.g., intravenous, intraarterial orsubcutaneous delivery) or local delivery (e.g., to the eye).

The endoglin polypeptides disclosed herein may be used in conjunction orsequentially with one or more additional therapeutic agents, including,for example, anti-angiogenesis agents, VEGF antagonists, anti-VEGFantibodies, anti-neoplastic compositions, cytotoxic agents,chemotherapeutic agents, anti-hormonal agents, and growth inhibitoryagents. Further examples of each of the foregoing categories ofmolecules are provided herein.

In certain aspects, the disclosure provides methods for inhibitingangiogenesis in a mammal by administering any of the endoglinpolypeptides described generally or specifically herein. The endoglinpolypeptide may be delivered locally (e.g., to the eye) or systemically(e.g., intravenously, intraarterially or subcutaneously). In certainembodiments, the disclosure provides a method for inhibitingangiogenesis in the eye of a mammal by administering an endoglinpolypeptide to the mammal at a location distal to the eye, e.g. bysystemic administration.

In certain aspects the disclosure provides methods for treating a tumorin a mammal. Such a method may comprise administering to a mammal thathas a tumor an effective amount of an endoglin polypeptide. A method mayfurther comprise administering one or more additional agents, including,for example, anti-angiogenesis agents, VEGF antagonists, anti-VEGFantibodies, anti-neoplastic compositions, cytotoxic agents,chemotherapeutic agents, anti-hormonal agents, and growth inhibitoryagents. A tumor may also be one that utilizes multiple pro-angiogenicfactors, such as a tumor that is resistant to anti-VEGF therapy.

In certain aspects, the disclosure provides methods for treatingpatients having a BMP9 or BMP10 related disorder. Examples of suchdisorders are provided herein, and may include, generally, disorders ofthe vasculature, hypertension, and fibrotic disorders.

In certain aspects the disclosure provides ophthalmic formulations. Suchformulations may comprise an endoglin polypeptide disclosed herein. Incertain aspects, the disclosure provides methods for treating anangiogenesis related disease of the eye. Such methods may compriseadministering systemically or to said eye a pharmaceutical formulationcomprising an effective amount of an endoglin polypeptide disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the native amino acid sequence of human ENG, isoform 1(L-ENG). The leader (residues 1-25) and predicted transmembrane domain(residues 587-611) are each underlined.

FIG. 2 shows the native nucleotide sequence encoding human ENG, isoform1 (L-ENG). Sequences encoding the leader (nucleotides 414-488) andpredicted transmembrane domain (nucleotides 2172-2246) are eachunderlined.

FIG. 3 shows the native amino acid sequence of human ENG, isoform 2(S-ENG). The leader (residues 1-25) and predicted transmembrane domain(residues 587-611) are each underlined. Compared to isoform 1, isoform 2has a shorter and distinct C-terminus, but the sequence of theextracellular domain (see FIG. 9) is identical.

FIG. 4 shows the native nucleotide sequence encoding human ENG, isoform2 (S-ENG). Sequences encoding the leader (nucleotides 414-488) andpredicted transmembrane domain (nucleotides 2172-2246) are eachunderlined.

FIG. 5 shows the native amino acid sequence of murine ENG, isoform 1(L-ENG). The leader (residues 1-26) and predicted transmembrane domain(residues 582-606) are underlined and bracket the extracellular domainof the mature peptide (see FIG. 10). Isoform 3 of murine ENG (GenBankaccession NM_(—)001146348) differs from the depicted sequence only inthe leader, where the threonine at position 23 (highlighted) is deletedand there is a glycine-to-serine substitution at position 24 (alsohighlighted).

FIG. 6 shows the native nucleotide sequence encoding murine ENG, isoform1 (L-ENG). Sequences encoding the leader (nucleotides 364-441) andpredicted transmembrane domain (nucleotides 2107-2181) are underlined.The nucleotide sequence encoding isoform 3 of murine ENG (GenBankaccession NM_(—)001146348) differs from the depicted sequence only inthe leader, specifically at positions 430-433 (highlighted).

FIG. 7 shows the native amino acid sequence of murine ENG, isoform 2(S-ENG). The leader (residues 1-26) and predicted transmembrane domain(residues 582-606) are underlined. Compared to isoform 1, isoform 2 hasa shorter and distinct C-terminus, but the sequence of the extracellulardomain (see FIG. 10) is identical.

FIG. 8 shows the native nucleotide sequence encoding murine ENG, isoform2 (S-ENG). Sequences encoding the leader (nucleotides 364-441) andpredicted transmembrane domain (nucleotides 2107-2181) are underlined.

FIG. 9 shows the amino acid sequence of the extracellular domain ofhuman ENG. The extracellular domains of the two human isoforms areidentical in both amino-acid and nucleotide sequence.

FIG. 10 shows the amino acid sequence of the extracellular domain ofmurine ENG, which is 69% identical to its human counterpart. Theextracellular domains of the two murine isoforms are identical in bothamino-acid and nucleotide sequence.

FIG. 11 shows an amino acid sequence of the human IgG1 Fc domain.Underlined residues are optional mutation sites as discussed in thetext.

FIG. 12 shows an N-terminally truncated amino acid sequence of the humanIgG1 Fc domain. Underlined residues are optional mutation sites asdiscussed in the text.

FIG. 13 shows the amino acid sequence of hENG(26-586)-hFc. The ENGdomain is underlined, the TPA leader sequence is double underlined, andlinker sequences are bold and highlighted.

FIG. 14 shows a nucleotide sequence encoding hENG(26-586)-hFc.Nucleotides encoding the ENG domain are underlined, those encoding theTPA leader sequence are double underlined, and those encoding linkersequences are bold and highlighted.

FIG. 15 shows the amino acid sequence of hENG(26-586)-hFc with anN-terminally truncated Fc domain. The ENG domain is underlined, the TPAleader sequence is double underlined, and linker sequences are bold andhighlighted.

FIG. 16 shows the amino acid sequence of mENG(27-581)-mFc. The ENGdomain is underlined, the TPA leader sequence is double underlined, andlinker sequences are bold and highlighted.

FIG. 17 shows a nucleotide sequence encoding mENG(27-581)-mFc.Nucleotides encoding the ENG domain are underlined, those encoding theTPA leader sequence are double underlined, and those encoding linkersequences are bold and highlighted.

FIG. 18 shows characterization of BMP-9 binding to hENG(26-586)-hFc, asdetermined in a surface plasmon resonance (SPR)-based assay. BMP-9binding to captured hENG(26-586)-hFc was assessed at ligandconcentrations of 0 and 0.01-0.625 nM (in two-fold increments, excluding0.3125 nM), and non-linear regression was used to determine the K_(D) as29 pM.

FIG. 19 shows characterization of BMP-10 binding to hENG(26-586)-hFc, asdetermined in an SPR-based assay. BMP-10 binding to capturedhENG(26-586)-hFc was assessed at ligand concentrations of 0 and0.01-1.25 nM (in two-fold increments), and non-linear regression wasused to determine the K_(D) as 400 pM.

FIG. 20 shows the effect of soluble human ENG extracellular domain,hENG(26-586), on binding of BMP-9 to ALK1. Concentrations ofhENG(26-586) from 0-50 nM were premixed with a fixed concentration ofBMP-9 (10 nM), and BMP-9 binding to captured ALK1 was determined by anSPR-based assay. The uppermost trace corresponds to no hENG(26-586),whereas the lowest trace corresponds to an ENG:BMP-9 ratio of 5:1.Binding of BMP-9 to ALK1 was inhibited by soluble hENG(26-586) in aconcentration-dependent manner with an IC₅₀ of 9.7 nM.

FIG. 21 shows the effect of soluble human ENG extracellular domain,hENG(26-586), on binding of BMP-10 to ALK1. Concentrations ofhENG(26-586) from 0-50 nM were premixed with a fixed concentration ofBMP-10 (10 nM), and BMP-10 binding to captured ALK1 was measured by anSPR-based assay. The uppermost trace corresponds to no hENG(26-586), andthe lowest trace corresponds to an ENG:BMP-10 ratio of 5:1. Binding ofBMP-10 to ALK1 was inhibited by soluble hENG(26-586) in aconcentration-dependent manner with an IC₅₀ of 6.3 nM.

FIG. 22 shows the effect of mENG(27-581)-hFC on cord formation by humanumbilical vein endothelial cells (HUVEC) in culture. Data are means ofduplicate cultures ±SD. The inducer endothelial cell growth substance(ECGS) doubled mean cord length compared to no treatment, andmENG(27-581)-hFc cut this increase by nearly 60%. In the absence ofstimulation (no treatment), mENG(27-581)-hFc had little effect.

FIG. 23 shows the effect of mENG(27-581)-hFc on VEGF-stimulatedangiogenesis in a chick chorioallantoic membrane (CAM) assay. Data aremeans±SEM; *, p<0.05. The number of additional blood vessels induced byVEGF treatment was decreased by 65% with concurrent mENG(27-581)-hFctreatment.

FIG. 24 shows the effect of mENG(27-581)-mFc treatment for 11 days onangiogenesis stimulated by a combination of the growth factors (GF)vascular endothelial growth factor (VEGF) and basic fibroblast growthfactor (FGF-2) in a mouse angioreactor assay. Angiogenesis in units ofrelative fluorescence ±SEM; *, p<0.05. mENG(27-581)-mFc completelyblocked GF-stimulated angiogenesis in this in vivo assay.

FIG. 25 shows the domain structure of hENG-Fc fusion constructs.Full-length ENG extracellular domain (residues 26-586 in top structure)consists of an orphan domain and N-terminal and C-terminal zonapellucida (ZP) domains. Below it are shown structures of selectedtruncated variants and whether they exhibit high-affinity binding (+/−)to BMP-9 and BMP-10 in an SPR-based assay.

FIG. 26 shows the amino acid sequence of hENG(26-437)-hFc. The ENGdomain is underlined, the TPA leader sequence is double underlined, andlinker sequences are bold and highlighted.

FIG. 27 shows a nucleotide sequence encoding hENG(26-437)-hFc.Nucleotides encoding the ENG domain are underlined, those encoding theTPA leader sequence are double underlined, and those encoding linkersequences are bold and highlighted.

FIG. 28 shows the amino acid sequence of hENG(26-378)-hFc with anN-terminally truncated Fc domain. The ENG domain is underlined, the TPAleader sequence is double underlined, and linker sequences are bold andhighlighted.

FIG. 29 shows a nucleotide sequence encoding hENG(26-378)-hFc with anN-terminally truncated Fc domain. Nucleotides encoding the ENG domainare underlined and those encoding linker sequences are bold andhighlighted.

FIG. 30 shows the amino acid sequence of hENG(26-359)-hFc. The ENGdomain is underlined, the TPA leader sequence is double underlined, andlinker sequences are bold and highlighted.

FIG. 31 shows a nucleotide sequence encoding hENG(26-359)-hFc.

Nucleotides encoding the ENG domain are underlined, those encoding theTPA leader sequence are double underlined, and those encoding linkersequences are bold and highlighted.

FIG. 32 shows the amino acid sequence of hENG(26-359)-hFc with anN-terminally truncated Fc domain. The ENG domain is underlined, the TPAleader sequence is double underlined, and linker sequences are bold andhighlighted.

FIG. 33 shows a nucleotide sequence encoding hENG(26-359)-hFc with anN-terminally truncated Fc domain. Nucleotides encoding the ENG domainare underlined, those encoding the TPA leader sequence are doubleunderlined, and those encoding linker sequences are bold andhighlighted.

FIG. 34 shows the amino acid sequence of hENG(26-346)-hFc with anN-terminally truncated Fc domain. The ENG domain is underlined, the TPAleader sequence is double underlined, and linker sequences are bold andhighlighted.

FIG. 35 shows a nucleotide sequence encoding hENG(26-346)-hFc with anN-terminally truncated Fc domain. Nucleotides encoding the ENG domainare underlined and those encoding linker sequences are bold andhighlighted.

FIG. 36 shows size-exclusion chromatograms for hENG(26-586)-hFc (A),hENG(26-359)-hFc (B), and hENG(26-346)-hFc (C) after the respectiveCHO-cell-derived proteins were purified by protein A affinitychromatography. Percent recovery of monomeric hENG(26-346)-hFc was equalto that of hENG(26-586)-hFc. In contrast, recovery of monomerichENG(26-359)-hFc was reduced by the presence of additionalhigh-molecular-weight aggregates, thus requiring additional proceduresto obtain purity equivalent to that of the other constructs.

FIG. 37 shows kinetic characterization of BMP-9 binding tohENG(26-586)-hFc (A), hENG(26-359)-hFc (B), and hENG(26-346)-hFc (C), asdetermined in an SPR-based assay. BMP-9 binding to capturedCHO-cell-derived proteins was assessed at ligand concentrations of0.0195-0.625 nM in two-fold increments. RU, response units. Note sloweroff-rates for the truncated variants compared to hENG(26-586)-hFc.

FIG. 38 shows the effect of hENG(26-359)-hFc on VEGF-stimulatedangiogenesis in a CAM assay. Data are means±SEM; *, p<0.05. The numberof additional blood vessels induced by VEGF treatment was decreased by75% with concurrent hENG(26-359)-hFc treatment, even thoughhENG(26-359)-hFc does not bind VEGF.

FIG. 39 shows the effect of hENG(26-346)-hFc treatment for 11 days onangiogenesis stimulated by a combination of the growth factors (GF) VEGFand FGF-2 in a mouse angioreactor assay. A. Angiogenesis in units ofrelative fluorescence ±SEM; *, p<0.05. B. Photographs of individualangioreactors (four per mouse) arranged by treatment group, with bloodvessel formation visible as darkened contents. Although unable to bindVEGF or FGF-2 itself, hENG(26-346)-hFc completely blocked GF-stimulatedangiogenesis in this in vivo assay.

FIG. 40 shows the effect of mENG(27-581)-mFc on growth of 4T1 mammarytumor xenografts in mice. Data are means±SEM. By day 24 postimplantation, tumor volume was 45% lower (p<0.05) in mice treated withmENG(27-581)-mFc compared to vehicle.

FIG. 41 shows the effect of mENG(27-581)-mFc on growth of Colon-26 tumorxenografts in mice. mENG(27-581)-mFc treatment inhibited tumor growth ina dose-dependent manner, with tumor volume in the high-dose group nearly70% lower than vehicle by day 58 post implantation.

DETAILED DESCRIPTION 1. Overview

In certain aspects, the present invention relates to ENG polypeptides.ENG (also known as CD105) is referred to as a coreceptor for thetransforming growth factor-β (TGF-β) superfamily of ligands and isimplicated in normal and pathological angiogenesis. ENG expression islow in quiescent vascular endothelium but upregulated in endothelialcells of healing wounds, developing embryos, inflammatory tissues, andsolid tumors (Dallas et al, 2008, Clin Cancer Res 14:1931-1937). Micehomozygous for null ENG alleles die early in gestation due to defectivevascular development (Li et al, 1999, Science 284:1534-1537), whereasheterozygous null ENG mice display angiogenic abnormalities as adults(Jerkic et al, 2006, Cardiovasc Res 69:845-854). In humans, ENG genemutations have been identified as the cause of hereditary hemorrhagictelangiectasia (Osler-Rendu-Weber syndrome) type-1 (HHT-1), an autosomaldominant form of vascular dysplasia characterized by arteriovenousmalformations resulting in direct flow (communication) from artery tovein (arteriovenous shunt) without an intervening capillary bed(McAllister et al, 1994, Nat Genet 8:345-351; Fernandez-L et al, 2006,Clin Med Res 4:66-78). Typical symptoms of patients with HHT includerecurrent epistaxis, gastrointestinal hemorrhage, cutaneous andmucocutaneous telangiectases, and arteriovenous malformations in thepulmonary, cerebral, or hepatic vasculature.

Although the specific role of ENG in angiogenesis remains to bedetermined, it is likely related to the prominent role of the TGF-βsignaling system in this process (Cheifetz et al, 1992, J Biol Chem267:19027-19030; Pardali et al, 2010, Trends Cell Biol 20:556-567).Significantly, ENG expression is upregulated in proliferating vascularendothelial cells within tumor tissues (Burrows et al, 1995, Clin CancerRes 1:1623-1634; Miller et al, 1999, Int J Cancer 81:568-572), and thenumber of ENG-expressing blood vessels in a tumor is negativelycorrelated with survival for a wide range of human tumors (Fonsatti etal, 2010, Cardiovasc Res 86:12-19). Thus, ENG is a promising target forantiangiogenic therapy generally, and for cancer in particular (Dallaset al, 2008, Clin Cancer Res 14:1931-1937; Bernabeu et al, 2009, BiochimBiophys Acta 1792:954-973).

Structurally, ENG is a homodimeric cell-surface glycoprotein. It belongsto the zona pelucida (ZP) family of proteins and consists of a shortC-terminal cytoplasmic domain, a single hydrophobic transmembranedomain, and a long extracellular domain (ECD) (Gougos et al, 1990, JBiol Chem 265:8361-8364). As determined by electron microscopy,monomeric ENG ECD consists of two ZP regions and an orphan domainlocated at the N-terminus (Llorca et al, 2007, J Mol Biol 365:694-705).In humans, alternative splicing of the primary transcript results in twoENG isoforms, one consisting of 658 residues (long, L, SEQ ID NO: 1) andthe other 625 residues (short, S, SEQ ID NO: 3), which differ only intheir cytoplasmic domain (Bellon et al, 1993, 23:2340-2345; ten Dijke etal, 2008, Angiogenesis 11:79-89). Murine ENG exists as three isoforms:L-ENG (SEQ ID NO: 5), S-ENG (SEQ ID NO: 7), and a third variant (isoform3) of unknown functional significance identical to L-ENG except forchanges at two positions within the leader sequence (Perez-Gomez et al,2005, Oncogene 24:4450-4461). The ECD of murine ENG displays 69% aminoacid identity with that of human ENG and lacks the Arg-Gly-Asp (RGD)integrin interaction motif found in the human protein. Recent evidencesuggests that the L-ENG and S-ENG isoforms may play different functionalroles in vivo (Blanco et al, 2008, Circ Res 103:1383-1392; ten Dijke etal, 2008, Angiogenesis 11:79-89).

As a coreceptor, ENG is thought to modulate responses of other receptorsto TGF-β family ligands without direct mediation of ligand signaling byitself. Ligands in the TGF-β family typically signal by binding to ahomodimeric type II receptor, which triggers recruitment andtransphosphorylation of a homodimeric type I receptor, thereby leadingto phosphorylation of Smad proteins responsible for transcriptionalactivation of specific genes (Massague, 2000, Nat Rev Mol Cell Biol1:169-178). Based on ectopic cellular expression assays, it has beenreported that ENG cannot bind ligands on its own and that its binding toTGF-β1, TGF-β3, activin A, bone morphogenetic protein-2 (BMP-2), andBMP-7 requires the presence of an appropriate type I and/or type IIreceptor (Barbara et al, 1999, J Biol Chem 274:584-594). Nevertheless,there is evidence that ENG expressed by a fibroblast cell line can bindTGF-β1 (St.-Jacques et al, 1994, Endocrinology 134:2645-2657), andrecent results in COS cells indicate that transfected full-length ENGcan bind BMP-9 in the absence of transfected type I or type II receptors(Scharpfenecker et al, 2007, J Cell Sci 120:964-972).

In addition to the foregoing, ENG can occur in a soluble form in vivounder certain conditions after proteolytic cleavage of the full-lengthmembrane-bound protein (Hawinkels et al, 2010, Cancer Res 70:4141-4150).Elevated levels of soluble ENG have been observed in the circulation ofpatients with cancer and preeclampsia (Li et al, 2000, Int J Cancer89:122-126; Calabro et al, 2003, J Cell Physiol 194:171-175; Venkateshaet al, 2006, Nat Med 12:642-649; Levine et al, 2006, N Engl J Med355:992-1005). Although the role of endogenous soluble ENG is poorlyunderstood, a protein corresponding to residues 26-437 of the ENGprecursor (amino acids 26-437 of SEQ ID NO: 1) has been proposed to actas a scavenger or trap for TGF-β family ligands (Venkatesha et al, 2006,Nat Med 12:642-649; WO-2007/143023), of which only TGF-β1 and TGF-β3have specifically been implicated.

The present disclosure relates to the discovery that polypeptidescomprising a truncated portion of the extracellular domain of ENG bindselectively to BMP9 and/or BMP10 and can act as BMP9 and/or BMP10antagonists, provide advantageous properties relative to the full-lengthextracellular domain, and may be used to inhibit angiogenesis mediatedby multiple angiogenic factors in vivo, including VEGF and basicfibroblast growth factor (FGF-2). In part, the disclosure provides theidentity of physiological, high-affinity ligands for soluble ENGpolypeptides. Surprisingly, soluble ENG polypeptides are shown herein tohave highly specific, high affinity binding for BMP-9 and BMP-10 whilenot exhibiting any meaningful binding to TGF-β1, TGF-β2 or TGF-β3, andmoreover, soluble ENG polypeptides are shown herein to inhibit BMP9 andBMP10 interaction with type II receptors, thereby inhibiting cellularsignal transduction. The disclosure further demonstrates that ENGpolypeptides inhibit angiogenesis. The data also demonstrate that an ENGpolypeptide can exert an anti-angiogenic effect despite the finding thatENG polypeptide does not exhibit meaningful binding to TGF-β1, TGF-β3,VEGF, or FGF-2.

Thus, in certain aspects, the disclosure provides endoglin polypeptidesas antagonists of BMP-9 or BMP-10 for use in inhibiting any BMP-9 orBMP-10 disorder generally, and particularly for inhibiting angiogenesis,including both VEGF-dependent angiogenesis and VEGF-independentangiogenesis. However, it should be noted that antibodies directed toENG itself are expected to have different effects from an ENGpolypeptide. A pan-neutralizing antibody against ENG (one that inhibitsthe binding of all strong and weak ligands) would be expected to inhibitthe signaling of such ligands through ENG but would not be expected toinhibit the ability of such ligands to signal through other receptors(e.g., ALK-1, ALK-2, BMPRII, ActRIIA or ActRIIB in the case of BMP-9 orBMP-10). It should further be noted that, given the existence of native,circulating soluble ENG polypeptides that, based on the data presentedhere, presumably act as natural BMP-9/10 antagonists, it is not clearwhether a neutralizing anti-ENG antibody would primarily inhibit themembrane bound form of ENG (thus acting as an ENG/BMP-9/10 antagonist)or the soluble form of ENG (thus acting as an ENG/BMP-9/10 agonist). Onthe other hand, based on this disclosure, an ENG polypeptide would beexpected to inhibit all of the ligands that it binds to tightly(including, for constructs such as those shown in the Examples, BMP-9 orBMP-10) but would not affect ligands that it binds to weakly. So, whilea pan-neutralizing antibody against ENG would block BMP-9 and BMP-10signaling through ENG, it would not block BMP-9 or BMP-10 signalingthrough another receptor. Also, while an ENG polypeptide may inhibitBMP-9 signaling through all receptors (including receptors besides ENG)it would not be expected to inhibit a weakly binding ligand signalingthrough any receptor, even ENG.

Proteins described herein are the human forms, unless otherwisespecified. Genbank references for the proteins are as follows: human ENGisoform 1 (L-ENG), NM_(—)001114753; human ENG isoform 2 (S-ENG),NM_(—)000118; murine ENG isoform 1 (L-ENG), NM_(—)007932; murine ENGisoform 2 (S-ENG), NM_(—)001146350; murine ENG isoform 3,NM_(—)001146348. Sequences of native ENG proteins from human and mouseare set forth in FIGS. 1-8.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussed inthe specification, to provide additional guidance to the practitioner indescribing the compositions and methods disclosed herein and how to makeand use them. The scope or meaning of any use of a term will be apparentfrom the specific context in which the term is used.

2. Soluble ENG Polypeptides

Except under certain conditions, naturally occurring ENG proteins aretransmembrane proteins, with a portion of the protein positioned outsidethe cell (the extracelluar portion) and a portion of the proteinpositioned inside the cell (the intracellular portion). Aspects of thepresent disclosure encompass polypeptides comprising a portion of theextracellular domain (ECD) of ENG.

In certain embodiments, the disclosure provides ENG polypeptides. ENGpolypeptides may include a polypeptide consisting of, or comprising, anamino acid sequence at least 90% identical, and optionally at least 95%,96%, 97%, 98%, 99%, or 100% identical to a truncated ECD domain of anaturally occurring ENG polypeptide, whose C-terminus occurs at any ofamino acids 333-378 of SEQ ID NO: 1 and which polypeptide does notinclude a sequence consisting of amino acids 379-430 of SEQ ID NO:1.Optionally, an ENG polypeptide does not include more than 5 consecutiveamino acids, or more than 10, 20, 30, 40, 50, 52, 60, 70, 80, 90, 100,150 or 200 or more consecutive amino acids from a sequence consisting ofamino acids 379-586 of SEQ ID NO: 1 or from a sequence consisting ofamino acids 379-581 of SEQ ID NO:1. The unprocessed ENG polypeptide mayeither include or exclude any signal sequence, as well as any sequenceN-terminal to the signal sequence. As elaborated herein, the N-terminusof the mature (processed) ENG polypeptide may occur at any of aminoacids 26-42 of SEQ ID NO: 1. Examples of mature ENG polypeptides includeamino acids 25-377 of SEQ ID NO: 23, amino acids 25-358 of SEQ ID NO:25, and amino acids 25-345 of SEQ ID NO: 29. Likewise, an ENGpolypeptide may comprise a polypeptide that is encoded by nucleotides73-1131 of SEQ ID NO: 24, nucleotides 73-1074 of SEQ ID NO: 26, ornucleotides 73-1035 of SEQ ID NO: 30, or silent variants thereof ornucleic acids that hybridize to the complement thereof under stringenthybridization conditions (generally, such conditions are known in theart but may, for example, involve hybridization in 50% v/v formamide,5×SSC, 2% w/v blocking agent, 0.1% N-lauroylsarcosine, and 0.3% SDS at65° C. overnight and washing in, for example, 5×SSC at about 65° C.).The term “ENG polypeptide” accordingly encompasses isolatedextracellular portions of ENG polypeptides, variants thereof (includingvariants that comprise, for example, no more than 2, 3, 4, 5, 10, 15,20, 25, 30, or 35 amino acid substitutions in the sequence correspondingto amino acids 26-378 of SEQ ID NO: 1), fragments thereof, and fusionproteins comprising any of the preceding, but in each case preferablyany of the foregoing ENG polypeptides will retain substantial affinityfor BMP-9 and/or BMP-10. Generally, an ENG polypeptide will be designedto be soluble in aqueous solutions at biologically relevanttemperatures, pH levels, and osmolarity.

Data presented here show that Fc fusion proteins comprising shorterC-terminally truncated variants of ENG polypeptides display noappreciable binding to TGF-β1 and TGF-β3 but instead display higheraffinity binding to BMP-9, with a markedly slower dissociation rate,compared to either ENG(26-437)-Fc or an Fc fusion protein comprising thefull-length ENG ECD. Specifically, C-terminally truncated variantsending at amino acids 378, 359, and 346 of SEQ ID NO: 1 were all foundto bind BMP-9 with substantially higher affinity (and to bind BMP-10with undiminished affinity) compared to ENG(26-437) or ENG(26-586).However, binding to BMP-9 and BMP-10 was completely disrupted by moreextensive C-terminal truncations to amino acids 332, 329, or 257. Thus,ENG polypeptides that terminate between amino acid 333 and amino acid378 are all expected to be active, but constructs ending at, or between,amino acids 346 and 359 may be most active. Forms ending at, or between,amino acids 360 and 378 are predicted to trend toward the intermediateligand binding affinity shown by ENG(26-378). Improvements in other keyparameters are expected with certain constructs ending at, or between,amino acids 333 and 378 based on improvements in protein expression andelimination half-life observed with ENG(26-346)-Fc compared to fusionproteins comprising full-length ENG ECD (see Examples). Any of thesetruncated variant forms may be desirable to use, depending on theclinical or experimental setting.

At the N-terminus, it is expected that an ENG polypeptide beginning atamino acid 26 (the initial glutamate), or before, of SEQ ID NO: 1 willretain ligand binding activity. As disclosed herein, an N-terminaltruncation to amino acid 61 of SEQ ID NO: 1 abolishes ligand binding, asdo more extensive N-terminal truncations. However, as also disclosedherein, consensus modeling of ENG primary sequences indicates thatordered secondary structure within the region defined by amino acids26-60 of SEQ ID NO: 1 is limited to a four-residue beta strand predictedwith high confidence at positions 42-45 of SEQ ID NO: 1 and atwo-residue beta strand predicted with very low confidence at positions28-29 of SEQ ID NO: 1. Thus, an active ENG polypeptide will begin at (orbefore) amino acid 26, preferentially, or at any of amino acids 27-42 ofSEQ ID NO: 1.

Taken together, an active portion of an ENG polypeptide may compriseamino acid sequences 26-333, 26-334, 26-335, 26-336, 26-337, 26-338,26-339, 26-340, 26-341, 26-342, 26-343, 26-344, 26-345, or 26-346 of SEQID NO: 1, as well as variants of these sequences starting at any ofamino acids 27-42 of SEQ ID NO: 1. Exemplary ENG polypeptides compriseamino acid sequences 26-346, 26-359, and 26-378 of SEQ ID NO: 1.Variants within these ranges are also contemplated, particularly thosehaving at least 80%, 85%, 90%, 95%, or 99% identity to the correspondingportion of SEQ ID NO: 1. An ENG polypeptide may not include the sequenceconsisting of amino acids 379-430 of SEQ ID NO:1.

As described above, the disclosure provides ENG polypeptides sharing aspecified degree of sequence identity or similarity to a naturallyoccurring ENG polypeptide. To determine the percent identity of twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The amino acid residues at corresponding amino acid positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue as the corresponding position in the second sequence,then the molecules are identical at that position (as used herein aminoacid “identity” is equivalent to amino acid “homology”). The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

In one embodiment, the percent identity between two amino acid sequencesis determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package (available at http://www.gcg.com). In aspecific embodiment, the following parameters are used in the GAPprogram: either a Blosum 62 matrix or a PAM250 matrix, and a gap weightof 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or6. In yet another embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)) (available at http://www.gcg.com). Exemplary parameters includeusing a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified,percent identity between two amino acid sequences is to be determinedusing the GAP program using a Blosum 62 matrix, a GAP weight of 10 and alength weight of 3, and if such algorithm cannot compute the desiredpercent identity, a suitable alternative disclosed herein should beselected.

In another embodiment, the percent identity between two amino acidsequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

Another embodiment for determining the best overall alignment betweentwo amino acid sequences can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.,6:237-245 (1990)). In a sequence alignment the query and subjectsequences are both amino acid sequences. The result of said globalsequence alignment is presented in terms of percent identity. In oneembodiment, amino acid sequence identity is performed using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci., 6:237-245 (1990)). In a specific embodiment, parametersemployed to calculate percent identity and similarity of an amino acidalignment comprise: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5 and Gap Size Penalty=0.05.

In certain embodiments, an ENG polypeptide binds to BMP-9 and BMP-10,and the ENG polypeptide does not show substantial binding to TGF-β1 orTGF-β3. Binding may be assessed using purified proteins in solution orin a surface plasmon resonance system, such as a Biacore™ system. ENGpolypeptides may be selected to exhibit an anti-angiogenic activity.Bioassays for angiogenesis inhibitory activity include the chickchorioallantoic membrane (CAM) assay, the mouse angioreactor assay, andassays for measuring the effect of administering isolated or synthesizedproteins on implanted tumors. The CAM assay, the mouse angioreactorassay, and other assays are described in the Examples.

ENG polypeptides may additionally include any of various leadersequences at the N-terminus. Such a sequence would allow the peptides tobe expressed and targeted to the secretion pathway in a eukaryoticsystem. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783 (1992).Alternatively, a native ENG signal sequence may be used to effectextrusion from the cell. Possible leader sequences include honeybeemellitin, TPA, and native leaders (SEQ ID NOs. 13-15, respectively).Examples of ENG-Fc fusion proteins incorporating a TPA leader sequenceinclude SEQ ID NOs: 23, 25, 27, and 29. Processing of signal peptidesmay vary depending on the leader sequence chosen, the cell type used andculture conditions, among other variables, and therefore actualN-terminal start sites for mature ENG polypeptides may shift by 1, 2, 3,4 or 5 amino acids in either the N-terminal or C-terminal direction.Examples of mature ENG-Fc fusion proteins include SEQ ID NOs: 33-36, asshown below with the ENG polypeptide portion underlined.

Human ENG(26-378)-hFc (truncated Fc) (SEQ ID NO: 33)ETVHCD LQPVGPERDE VTYTTSQVSK GCVAQAPNAI LEVHVLFLEFPTGPSQLELT LQASKQNGTW PREVLLVLSV NSSVFLHLQALGIPLHLAYN SSLVTFQEPP GVNTTELPSF PKTQILEWAAERGPITSAAE LNDPQSILLR LGQAQGSLSF CMLEASQDMGRTLEWRPRTP ALVRGCHLEG VAGHKEAHIL RVLPGHSAGPRTVTVKVELS CAPGDLDAVL ILQGPPYVSW LIDANHNMQIWTTGEYSFKI FPEKNIRGFK LPDTPQGLLG EARMLNASIVASFVELPLAS IVSLHASSCG GRLQTSPAPI QTTPPKDTCSPELLMSLIQT KCADDAMTLV LKKELVATGG GTHTCPPCPAPELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNYKTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKHuman ENG(26-359)-hFc (SEQ ID NO: 34)ETVHCD LQPVGPERDE VTYTTSQVSK GCVAQAPNAI LEVHVLFLEFPTGPSQLELT LQASKQNGTW PREVLLVLSV NSSVFLHLQALGIPLHLAYN SSLVTFQEPP GVNTTELPSF PKTQILEWAAERGPITSAAE LNDPQSILLR LGQAQGSLSF CMLEASQDMGRTLEWRPRTP ALVRGCHLEG VAGHKEAHIL RVLPGHSAGPRTVTVKVELS CAPGDLDAVL ILQGPPYVSW LIDANHNMQIWTTGEYSFKI FPEKNIRGFK LPDTPQGLLG EARMLNASIVASFVELPLAS IVSLHASSCG GRLQTSPAPI QTTPPKDTCSPELLMSLITG GGPKSCDKTH TCPPCPAPEL LGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEVHNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVSNKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVSLTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSFFLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKHuman ENG(26-359)-hFc (truncated Fc) (SEQ ID NO: 35)ETVHCD LQPVGPERDE VTYTTSQVSK GCVAQAPNAI LEVHVLFLEFPTGPSQLELT LQASKQNGTW PREVLLVLSV NSSVFLHLQALGIPLHLAYN SSLVTFQEPP GVNTTELPSF PKTQILEWAAERGPITSAAE LNDPQSILLR LGQAQGSLSF CMLEASQDMGRTLEWRPRTP ALVRGCHLEG VAGHKEAHIL RVLPGHSAGPRTVTVKVELS CAPGDLDAVL ILQGPPYVSW LIDANHNMQIWTTGEYSFKI FPEKNIRGFK LPDTPQGLLG EARMLNASIVASFVELPLAS IVSLHASSCG GRLQTSPAPI QTTPPKDTCSPELLMSLITG GGTHTCPPCP APELLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTKPREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKLTVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKHuman ENG(26-346)-hFc (truncated Fc) (SEQ ID NO: 36)ETVHCD LQPVGPERDE VTYTTSQVSK GCVAQAPNAI LEVHVLFLEFPTGPSQLELT LQASKQNGTW PREVLLVLSV NSSVFLHLQALGIPLHLAYN SSLVTFQEPP GVNTTELPSF PKTQILEWAAERGPITSAAE LNDPQSILLR LGQAQGSLSF CMLEASQDMGRTLEWRPRTP ALVRGCHLEG VAGHKEAHIL RVLPGHSAGPRTVTVKVELS CAPGDLDAVL ILQGPPYVSW LIDANHNMQIWTTGEYSFKI FPEKNIRGFK LPDTPQGLLG EARMLNASIVASFVELPLAS IVSLHASSCG GRLQTSPAPI QTTPPTGGGTHTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVVVDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

In certain embodiments, the present disclosure contemplates specificmutations of the ENG polypeptides so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (orasparagines-X-serine) (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type ENG polypeptide (for O-linked glycosylation sites). A varietyof amino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Another means of increasing thenumber of carbohydrate moieties on an ENG polypeptide is by chemical orenzymatic coupling of glycosides to the ENG polypeptide. Depending onthe coupling mode used, the sugar(s) may be attached to (a) arginine andhistidine; (b) free carboxyl groups; (c) free sulfhydryl groups such asthose of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp.259-306, incorporated by reference herein. Removal of one or morecarbohydrate moieties present on an ENG polypeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the ENG polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ENG polypeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al. (1987) Meth.Enzymol. 138:350. The sequence of an ENG polypeptide may be adjusted, asappropriate, depending on the type of expression system used, asmammalian, yeast, insect and plant cells may all introduce differingglycosylation patterns that can be affected by the amino acid sequenceof the peptide. In general, ENG polypeptides for use in humans will beexpressed in a mammalian cell line that provides proper glycosylation,such as HEK293 or CHO cell lines, although other mammalian expressioncell lines, yeast cell lines with engineered glycosylation enzymes, andinsect cells are expected to be useful as well.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of an ENG polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, ENG polypeptide variants which can act as either agonistsor antagonist, or alternatively, which possess novel activities alltogether. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ENG polypeptidevariant may be screened for ability to bind to an ENG ligand, to preventbinding of an ENG ligand to an ENG polypeptide or to interfere withsignaling caused by an ENG ligand. The activity of an ENG polypeptide orits variants may also be tested in a cell-based or in vivo assay,particularly any of the assays disclosed in the Examples.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to an ENG polypeptide comprisingan extracellular domain of a naturally occurring ENG polypeptide.Likewise, mutagenesis can give rise to variants which have serumhalf-lives dramatically different than the corresponding wild-type ENGpolypeptide. For example, the altered protein can be rendered eithermore stable or less stable to proteolytic degradation or other processeswhich result in destruction of, or otherwise elimination or inactivationof, a native ENG polypeptide. Such variants, and the genes which encodethem, can be utilized to alter ENG polypeptide levels by modulating thehalf-life of the ENG polypeptides. For instance, a short half-life cangive rise to more transient biological effects and can allow tightercontrol of recombinant ENG polypeptide levels within the patient. In anFc fusion protein, mutations may be made in the linker (if any) and/orthe Fc portion to alter the half-life of the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ENG polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ENG polypeptidenucleotide sequences are expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay).

There are many ways by which the library of potential ENG polypeptidevariants can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then be ligatedinto an appropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic AcidRes. 11:477). Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al., (1990)Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433;Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNASUSA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ENG polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ENG polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ENG polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include ENG ligand binding assays and ligand-mediated cellsignaling assays.

In certain embodiments, the ENG polypeptides of the disclosure mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ENG polypeptides. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, pegylation (polyehthyleneglycol) and acylation. As a result, the modified ENG polypeptides maycontain non-amino acid elements, such as polyethylene glycols, lipids,poly- or mono-saccharide, and phosphates. Effects of such non-amino acidelements on the functionality of an ENG polypeptide may be tested asdescribed herein for other ENG polypeptide variants. When an ENGpolypeptide is produced in cells by cleaving a nascent form of the ENGpolypeptide, post-translational processing may also be important forcorrect folding and/or function of the protein. Different cells (such asCHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the ENG polypeptides.

In certain aspects, functional variants or modified forms of the ENGpolypeptides include fusion proteins having at least a portion of theENG polypeptides and one or more fusion domains. Well known examples ofsuch fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress™ system (Qiagen) useful with(HIS₆) fusion partners. As another example, a fusion domain may beselected so as to facilitate detection of the ENG polypeptides. Examplesof such detection domains include the various fluorescent proteins(e.g., GFP) as well as “epitope tags,” which are usually short peptidesequences for which a specific antibody is available. Well known epitopetags for which specific monoclonal antibodies are readily availableinclude FLAG, influenza virus haemagglutinin (HA), and c-myc tags. Insome cases, the fusion domains have a protease cleavage site, such asfor Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain preferred embodiments, an ENG polypeptide isfused with a domain that stabilizes the ENG polypeptide in vivo (a“stabilizer” domain). By “stabilizing” is meant anything that increasesserum half life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains.

As specific examples, the present disclosure provides fusion proteinscomprising variants of ENG polypeptides fused to one of two Fc domainsequences (e.g., SEQ ID NOs: 11, 12). Optionally, the Fc domain has oneor more mutations at residues such as Asp-265, Lys-322, and Asn-434(numbered in accordance with the corresponding full-length IgG). Incertain cases, the mutant Fc domain having one or more of thesemutations (e.g., Asp-265 mutation) has reduced ability of binding to theFey receptor relative to a wildtype Fc domain. In other cases, themutant Fc domain having one or more of these mutations (e.g., Asn-434mutation) has increased ability of binding to the MHC class I-relatedFc-receptor (FcRN) relative to a wildtype Fc domain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ENG polypeptide may be placed C-terminalto a heterologous domain, or, alternatively, a heterologous domain maybe placed C-terminal to an ENG polypeptide. The ENG polypeptide domainand the heterologous domain need not be adjacent in a fusion protein,and additional domains or amino acid sequences may be included C- orN-terminal to either domain or between the domains.

As used herein, the term “immunoglobulin Fc domain” or simply “Fc” isunderstood to mean the carboxyl-terminal portion of an immunoglobulinchain constant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. For example, an immunoglobulin Fc regionmay comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2domain and a CH3 domain, or 5) a combination of two or more domains andan immunoglobulin hinge region. In a preferred embodiment theimmunoglobulin Fc region comprises at least an immunoglobulin hingeregion a CH2 domain and a CH3 domain, and preferably lacks the CH1domain.

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4). Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) andIgM (Igμ), may be used. The choice of appropriate immunoglobulin heavychain constant region is discussed in detail in U.S. Pat. Nos.5,541,087, and 5,726,044. The choice of particular immunoglobulin heavychain constant region sequences from certain immunoglobulin classes andsubclasses to achieve a particular result is considered to be within thelevel of skill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH₃ domain of Fcgamma or the homologous domains in any of IgA, IgD, IgE, or IgM.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the methods and compositions disclosed herein.One example would be to introduce amino acid substitutions in the upperCH2 region to create an Fc variant with reduced affinity for Fcreceptors (Cole et al. (1997) J. Immunol. 159:3613).

In certain embodiments, the present disclosure makes available isolatedand/or purified forms of the ENG polypeptides, which are isolated from,or otherwise substantially free of (e.g., at least 80%, 90%, 95%, 96%,97%, 98%, or 99% free of), other proteins and/or other ENG polypeptidespecies. ENG polypeptides will generally be produced by expression fromrecombinant nucleic acids.

In certain embodiments, the disclosure includes nucleic acids encodingsoluble ENG polypeptides comprising the coding sequence for anextracellular portion of an ENG protein. In further embodiments, thisdisclosure also pertains to a host cell comprising such nucleic acids.The host cell may be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present disclosure may be expressed in bacterialcells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art. Accordingly, some embodiments ofthe present disclosure further pertain to methods of producing the ENGpolypeptides. It has been established that ENG-Fc fusion proteins setforth in SEQ ID NOs: 25 and 29 and expressed in CHO cells have potentanti-angiogenic activity.

3. Nucleic Acids Encoding ENG Polypeptides

In certain aspects, the disclosure provides isolated and/or recombinantnucleic acids encoding any of the ENG polypeptides, including fragments,functional variants and fusion proteins disclosed herein. For example,SEQ ID NOs: 2 and 4 encode long and short isoforms, respectively, of thenative human ENG precursor polypeptide, whereas SEQ ID NO: 30 encodesone variant of ENG extracellular domain fused to an IgG1 Fc domain. Thesubject nucleic acids may be single-stranded or double stranded. Suchnucleic acids may be DNA or RNA molecules. These nucleic acids may beused, for example, in methods for making ENG polypeptides or as directtherapeutic agents (e.g., in an antisense, RNAi or gene therapyapproach).

In certain aspects, the subject nucleic acids encoding ENG polypeptidesare further understood to include nucleic acids that are variants of SEQID NOs: 24, 26, 28, or 30. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NOs: 24, 26, 28, or 30. One ofordinary skill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NOs: 24, 26, 28, or 30, and variants of SEQ IDNOs: 24, 26, 28, or 30 are also within the scope of this disclosure. Infurther embodiments, the nucleic acid sequences of the disclosure can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence, or in a DNA library.

In other embodiments, nucleic acids of the disclosure also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequences designated in SEQ ID NOs: 24, 26, 28, or 30,complement sequences of SEQ ID NOs: 24, 26, 28, or 30, or fragmentsthereof. As discussed above, one of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 24, 26, 28, or 30 due to degeneracy in the genetic codeare also within the scope of the disclosure. For example, a number ofamino acids are designated by more than one triplet. Codons that specifythe same amino acid, or synonyms (for example, CAU and CAC are synonymsfor histidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this disclosure.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects disclosed herein, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ENG polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ENG polypeptide. Accordingly, theterm regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ENG polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid included in the disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant ENG polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ENG polypeptides in CHO cells, such as a Pcmv-Script vector(Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad,Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As will beapparent, the subject gene constructs can be used to cause expression ofthe subject ENG polypeptides in cells propagated in culture, e.g., toproduce proteins, including fusion proteins or variant proteins, forpurification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NOs: 24, 26,28, or 30) for one or more of the subject ENG polypeptides. The hostcell may be any prokaryotic or eukaryotic cell. For example, an ENGpolypeptide disclosed herein may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject ENG polypeptides. For example, a host celltransfected with an expression vector encoding an ENG polypeptide can becultured under appropriate conditions to allow expression of the ENGpolypeptide to occur. The ENG polypeptide may be secreted and isolatedfrom a mixture of cells and medium containing the ENG polypeptide.Alternatively, the ENG polypeptide may be retained cytoplasmically or ina membrane fraction and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The subject ENG polypeptides can be isolated from cell culture medium,host cells, or both, using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, immunoaffinitypurification with antibodies specific for particular epitopes of the ENGpolypeptides and affinity purification with an agent that binds to adomain fused to the ENG polypeptide (e.g., a protein A column may beused to purify an ENG-Fc fusion). In a preferred embodiment, the ENGpolypeptide is a fusion protein containing a domain which facilitatesits purification. As an example, purification may be achieved by aseries of column chromatography steps, including, for example, three ormore of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ENGpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ENG polypeptide (e.g., see Hochuliet al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

Examples of categories of nucleic acid compounds that are antagonists ofENG, BMP-9, or BMP-10 include antisense nucleic acids, RNAi constructsand catalytic nucleic acid constructs. A nucleic acid compound may besingle or double stranded. A double stranded compound may also includeregions of overhang or non-complementarity, where one or the other ofthe strands is single stranded. A single stranded compound may includeregions of self-complementarity, meaning that the compound forms aso-called “hairpin” or “stem-loop” structure, with a region of doublehelical structure. A nucleic acid compound may comprise a nucleotidesequence that is complementary to a region consisting of no more than1000, no more than 500, no more than 250, no more than 100 or no morethan 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length ENGnucleic acid sequence or ligand nucleic acid sequence. The region ofcomplementarity will preferably be at least 8 nucleotides, andoptionally at least 10 or at least 15 nucleotides, and optionallybetween 15 and 25 nucleotides. A region of complementarity may fallwithin an intron, a coding sequence, or a noncoding sequence of thetarget transcript, such as the coding sequence portion. Generally, anucleic acid compound will have a length of about 8 to about 500nucleotides or base pairs in length, and optionally the length will beabout 14 to about 50 nucleotides. A nucleic acid may be a DNA(particularly for use as an antisense), RNA, or RNA:DNA hybrid. Any onestrand may include a mixture of DNA and RNA, as well as modified formsthat cannot readily be classified as either DNA or RNA Likewise, adouble stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any onestrand may also include a mixture of DNA and RNA, as well as modifiedforms that cannot readily be classified as either DNA or RNA. A nucleicacid compound may include any of a variety of modifications, includingone or modifications to the backbone (the sugar-phosphate portion in anatural nucleic acid, including internucleotide linkages) or the baseportion (the purine or pyrimidine portion of a natural nucleic acid). Anantisense nucleic acid compound will preferably have a length of about15 to about 30 nucleotides and will often contain one or moremodifications to improve characteristics such as stability in the serum,in a cell or in a place where the compound is likely to be delivered,such as the stomach in the case of orally delivered compounds and thelung for inhaled compounds. In the case of an RNAi construct, the strandcomplementary to the target transcript will generally be RNA ormodifications thereof. The other strand may be RNA, DNA, or any othervariation. The duplex portion of double stranded or single stranded“hairpin” RNAi construct will preferably have a length of 18 to 40nucleotides in length and optionally about 21 to 23 nucleotides inlength, so long as it serves as a Dicer substrate. Catalytic orenzymatic nucleic acids may be ribozymes or DNA enzymes and may alsocontain modified forms. Nucleic acid compounds may inhibit expression ofthe target by about 50%, 75%, 90%, or more when contacted with cellsunder physiological conditions and at a concentration where a nonsenseor sense control has little or no effect. Preferred concentrations fortesting the effect of nucleic acid compounds are 1, 5 and 10 micromolar.Nucleic acid compounds may also be tested for effects on, for example,angiogenesis.

4. Alterations in Fc-Fusion Proteins

The application further provides ENG-Fc fusion proteins with engineeredor variant Fc regions. Such antibodies and Fc fusion proteins may beuseful, for example, in modulating effector functions, such as,antigen-dependent cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC). Additionally, the modifications may improve thestability of the antibodies and Fc fusion proteins. Amino acid sequencevariants of the antibodies and Fc fusion proteins are prepared byintroducing appropriate nucleotide changes into the DNA, or by peptidesynthesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the antibodies and Fc fusion proteins disclosed herein. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the antibodies and Fc fusion proteins,such as changing the number or position of glycosylation sites.

Antibodies and Fc fusion proteins with reduced effector function may beproduced by introducing changes in the amino acid sequence, including,but are not limited to, the Ala-Ala mutation described by Bluestone etal. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 CellImmunol 200; 16-26). Thus in certain embodiments, antibodies and Fcfusion proteins of the disclosure with mutations within the constantregion including the Ala-Ala mutation may be used to reduce or abolisheffector function. According to these embodiments, antibodies and Fcfusion proteins may comprise a mutation to an alanine at position 234 ora mutation to an alanine at position 235, or a combination thereof. Inone embodiment, the antibody or Fc fusion protein comprises an IgG4framework, wherein the Ala-Ala mutation would describe a mutation(s)from phenylalanine to alanine at position 234 and/or a mutation fromleucine to alanine at position 235. In another embodiment, the antibodyor Fc fusion protein comprises an IgG1 framework, wherein the Ala-Alamutation would describe a mutation(s) from leucine to alanine atposition 234 and/or a mutation from leucine to alanine at position 235.The antibody or Fc fusion protein may alternatively or additionallycarry other mutations, including the point mutation K322A in the CH2domain (Hezareh et al. 2001 J Virol. 75: 12161-8).

In particular embodiments, the antibody or Fc fusion protein may bemodified to either enhance or inhibit complement dependent cytotoxicity(CDC). Modulated CDC activity may be achieved by introducing one or moreamino acid substitutions, insertions, or deletions in an Fc region (see,e.g., U.S. Pat. No. 6,194,551). Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved or reduced internalizationcapability and/or increased or decreased complement-mediated cellkilling. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes,B. J. Immunol. 148:2918-2922 (1992), WO99/51642, Duncan & Winter Nature322: 738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821;and WO94/29351.

5. Therapeutic Uses

The disclosure provides methods and compositions for treating orpreventing conditions of dysregulated angiogenesis, including bothneoplastic and non-neoplastic disorders. Also provided are methods andcompositions for treating or preventing certain cardiovasculardisorders. In addition, the disclosure provides methods and compositionsfor treating or preventing fibrotic disorders and conditions. Inaddition the disclosure provides methods for treating disordersassociated with BMP9 and/or BMP10 activity.

The disclosure provides methods of inhibiting angiogenesis in a mammalby administering to a subject an effective amount of a an ENGpolypeptide, including an ENG-Fc fusion protein or nucleic acidantagonists (e.g., antisense or siRNA) of the foregoing, hereaftercollectively referred to as “therapeutic agents”. The data presentedindicate specifically that the anti-angiogenic therapeutic agentsdisclosed herein may be used to inhibit tumor-associated angiogenesis.It is expected that these therapeutic agents will also be useful ininhibiting angiogenesis in the eye.

Angiogenesis-associated diseases include, but are not limited to,angiogenesis-dependent cancer, including, for example, solid tumors,blood born tumors such as leukemias, and tumor metastases; benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; andangiofibroma.

In particular, polypeptide therapeutic agents of the present disclosureare useful for treating or preventing a cancer (tumor), and particularlysuch cancers as are known to rely on angiogenic processes to supportgrowth. Unlike most anti-angiogenic agents, ENG polypeptides affectangiogenesis induced by multiple factors. This is highly relevant incancers, where a cancer will frequently acquire multiple factors thatsupport tumor angiogenesis. Thus, the therapeutic agents disclosedherein will be particularly effective in treating tumors that areresistant to treatment with a drug that targets a single angiogenicfactor (e.g., bevacizumab, which targets VEGF), and may also beparticularly effective in combination with other anti-angiogeniccompounds that work by a different mechanism.

Dysregulation of angiogenesis can lead to many disorders that can betreated by compositions and methods of the invention. These disordersinclude both neoplastic and non-neoplastic conditions. The terms“cancer” and “cancerous” refer to, or describe, the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth/proliferation. Examples of cancer, or neoplastic disorders,include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma, gastric cancer, melanoma, and various typesof head and neck cancer, including squamous cell head and neck cancer.Other examples of neoplastic disorders and related conditions includeesophageal carcinomas, thecomas, arrhenoblastomas, endometrialhyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi'ssarcoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, Wilm'stumor, renal cell carcinoma, prostate carcinoma, abnormal vascularproliferation associated with phakomatoses, and Meigs' syndrome. Acancer that is particularly amenable to treatment with the therapeuticagents described herein may be characterized by one or more of thefollowing: the cancer has angiogenic activity, elevated ENG levelsdetectable in the tumor or the serum, increased BMP-9 or BMP-10expression levels or biological activity, is metastatic or at risk ofbecoming metastatic, or any combination thereof.

Non-neoplastic disorders with dysregulated angiogenesis that areamenable to treatment with ENG polypeptides useful in the inventioninclude, but are not limited to, undesired or aberrant hypertrophy,arthritis, rheumatoid arthritis, psoriasis, psoriatic plaques,sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic andother proliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, diabetic macular edema, corneal neovascularization,corneal graft neovascularization, corneal graft rejection,retinal/choroidal neovascularization, neovascularization of the angle(rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis, refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), hemophilic joints, hypertrophic scars, inhibition of hairgrowth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion. Further examples ofsuch disorders include an epithelial or cardiac disorder.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject methods of the disclosure can beused alone. Alternatively, the subject methods may be used incombination with other conventional anti-cancer therapeutic approachesdirected to treatment or prevention of proliferative disorders (e.g.,tumor). For example, such methods can be used in prophylactic cancerprevention, prevention of cancer recurrence and metastases aftersurgery, and as an adjuvant of other conventional cancer therapy. Thepresent disclosure recognizes that the effectiveness of conventionalcancer therapies (e.g., chemotherapy, radiation therapy, phototherapy,immunotherapy, and surgery) can be enhanced through the use of a subjectpolypeptide therapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a therapeutic agent disclosed herein is administered in combinationwith another conventional anti-neoplastic agent, either concomitantly orsequentially, such therapeutic agent may enhance the therapeutic effectof the anti-neoplastic agent or overcome cellular resistance to suchanti-neoplastic agent. This allows decrease of dosage of ananti-neoplastic agent, thereby reducing the undesirable side effects, orrestores the effectiveness of an anti-neoplastic agent in resistantcells.

According to the present disclosure, the antiangiogenic agents describedherein may be used in combination with other compositions and proceduresfor the treatment of diseases. For example, a tumor may be treatedconventionally with surgery, radiation or chemotherapy combined with theENG polypeptide, and then the ENG polypeptide may be subsequentlyadministered to the patient to extend the dormancy of micrometastasesand to stabilize any residual primary tumor.

Many anti-angiogenesis agents have been identified and are known in thearts, including those listed herein and, e.g., listed by Carmeliet andJain, Nature 407:249-257 (2000); Ferrara et al., Nature Reviews:DrugDiscovery, 3:391-400 (2004); and Sato Int. J. Clin. Oncol, 8:200-206(2003). See also, US Patent Application US20030055006. In oneembodiment, an ENG polypeptide is used in combination with an anti-VEGFneutralizing antibody (or fragment) and/or another VEGF antagonist or aVEGF receptor antagonist including, but not limited to, for example,soluble VEGF receptor (e.g., VEGFR-I, VEGFR-2, VEGFR-3, neuropillins(e.g., NRP1, NRP2)) fragments, aptamers capable of blocking VEGF orVEGFR, neutralizing anti-VEGFR antibodies, low molecule weightinhibitors of VEGFR tyrosine kinases (RTK), antisense strategies forVEGF, ribozymes against VEGF or VEGF receptors, antagonist variants ofVEGF; and any combinations thereof. Alternatively, or additionally, twoor more angiogenesis inhibitors may optionally be co-administered to thepatient in addition to VEGF antagonist and other agent. In certainembodiment, one or more additional therapeutic agents, e.g., anti-canceragents, can be administered in combination with an ENG polypeptide, theVEGF antagonist, and an anti-angiogenesis agent.

The terms “VEGF” and “VEGF-A” are used interchangeably to refer to the165-amino acid vascular endothelial cell growth factor and related 121-,145-, 183-, 189-, and 206-amino acid vascular endothelial cell growthfactors, as described by Leung et al. Science, 246:1306 (1989), Houck etal. Mol Endocrinol, 5:1806 (1991), and, Robinson & Stringer, J Cell Sci,144(5):853-865 (2001), together with the naturally occurring allelic andprocessed forms thereof.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including its binding to one or more VEGF receptors. VEGFantagonists include anti-VEGF antibodies and antigen-binding fragmentsthereof, receptor molecules and derivatives which bind specifically toVEGF thereby sequestering its binding to one or more receptors,anti-VEGF receptor antibodies and VEGF receptor antagonists such assmall molecule inhibitors of the VEGFR tyrosine kinases, and fusionsproteins, e.g., VEGF-Trap (Regeneron), VEGF121-gelonin (Peregrine). VEGFantagonists also include antagonist variants of VEGF, antisensemolecules directed to VEGF, RNA aptamers, and ribozymes against VEGF orVEGF receptors.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. The anti-VEGF antibody can be usedas a therapeutic agent in targeting and interfering with diseases orconditions wherein the VEGF activity is involved. See, e.g., U.S. Pat.Nos. 6,582,959, 6,703,020; WO98/45332; WO 96/30046; WO94/10202,WO2005/044853; EP 0666868B1; US Patent Applications 20030206899,20030190317, 20030203409, 20050112126, 20050186208, and 20050112126;Popkov et al, Journal of Immunological Methods 288:149-164 (2004); andWO2005012359. An anti-VEGF antibody will usually not bind to other VEGFhomologues such as VEGF-B or VEGF-C, nor other growth factors such asPlGF, PDGF or bFGF. The anti-VEGF antibody “Bevacizumab (BV)”, alsoknown as “rhuMAb VEGF” or “Avastin®”, is a recombinant humanizedanti-VEGF monoclonal antibody generated according to Presta et al.Cancer Res. 57:4593-4599 (1997). It comprises mutated human IgG1framework regions and antigen-binding complementarity-determiningregions from the murine anti-hVEGF monoclonal antibody A.4.6.1 thatblocks binding of human VEGF to its receptors. Approximately 93% of theamino acid sequence of Bevacizumab, including most of the frameworkregions, is derived from human IgG1, and about 7% of the sequence isderived from the murine antibody A4.6.1. Bevacizumab has a molecularmass of about 149,000 daltons and is glycosylated. Bevacizumab and otherhumanized anti-VEGF antibodies, including the anti-VEGF antibodyfragment “ranibizumab”, also known as “Lucentis®”, are further describedin U.S. Pat. No. 6,884,879 issued Feb. 26, 2005.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent”. Examples of therapeutic agents (anti-cancer agents,also termed “anti-neoplastic agent” herein) include, but are not limitedto, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxicagents, agents used in radiation therapy, anti-angiogenesis agents,apoptotic agents, anti-tubulin agents, toxins, and other-agents to treatcancer, e.g., anti-VEGF neutralizing antibody, VEGF antagonist,anti-HER-2, anti-CD20, an epidermal growth factor receptor (EGFR)antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor,erlotinib, a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe ErbB2, ErbB3, ErbB4, or VEGF receptor(s), inhibitors for receptortyrosine kinases for platet-derived growth factor (PDGF) and/or stemcell factor (SCF) (e.g., imatinib mesylate (Gleevec® Novartis)),TRAIL/Apo2L, and other bioactive and organic chemical agents, etc.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promotes angiogenesis, endothelialcell growth, stability of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, P1GF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, ANGPTL3,ALK-1, etc. It would also include factors that accelerate wound healing,such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF,epidermal growth factor (EGF), CTGF and members of its family, and TGF-αand TGF-β. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol,53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003);Ferrara & Alitalo, Nature Medicine 5(12): 1359-1364 (1999); Tonini etal., Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listing angiogenicfactors); and, Sato Int. J. Clin. Oncol., 8:200-206 (2003).

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide (including, e.g., aninhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, arecombinant protein, an antibody, or conjugates or fusion proteinsthereof, that inhibits angiogenesis, vasculogenesis, or undesirablevascular permeability, either directly or indirectly. For example, ananti-angiogenesis agent is an antibody or other antagonist to anangiogenic agent as defined above, e.g., antibodies to VEGF, antibodiesto VEGF receptors, small molecules that block VEGF receptor signaling(e.g., PTK787/ZK2284, SU6668, SUTENT®/SU 11248 (sunitinib malate),AMG706, or those described in, e.g., international patent application WO2004/113304). Anti-angiogensis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore, Annu. Rev. Physiol, 53:217-39 (1991); Streit and Detmar,Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenictherapy in malignant melanoma); Ferrara & Alitalo, Nat Med 5(12):1359-1364 (1999); Tonini et al, Oncogene, 22:6549-6556 (2003) (e.g.,Table 2 listing antiangiogenic factors); and, Sato Int. J. Clin. Oncol,8:200-206 (2003) (e.g., Table 1 lists Anti-angiogenesis agents used inclinical trials).

In certain aspects of the invention, other therapeutic agents useful forcombination tumor therapy with an ENG polypeptide include other cancertherapies: e.g., surgery, cytoxic agents, radiological treatmentsinvolving irradiation or administration of radioactive substances,chemotherapeutic agents, anti-hormonal agents, growth inhibitory agents,anti-neoplastic compositions, and treatment with anti-cancer agentslisted herein and known in the art, or combinations thereof.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gammall and calicheamicin omegall (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY1 17018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In addition,such definition of chemotherapeutic agents includes bisphosphonates suchas clodronate (for example, BONEFOS® or OSTAC®), DIDROC AL® etidronate,NE-58095, ZOMET A® zoledronic acid/zoledronate, FOSAMAX® alendronate,AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);antisense oligonucleotides, particularly those that inhibit expressionof genes in signaling pathways implicated in abherant cellproliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as THERATOPE®vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine,LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1inhibitor; ABARELIX® rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dualtyrosine kinase small-molecule inhibitor also known as GW572016); andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce Gl arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest Gl also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

Angiogenesis-inhibiting agents can also be given prophylactically toindividuals known to be at high risk for developing new or re-currentcancers. Accordingly, an aspect of the disclosure encompasses methodsfor prophylactic prevention of cancer in a subject, comprisingadministrating to the subject an effective amount of an ENG polypeptideand/or a derivative thereof, or another angiogenesis-inhibiting agent ofthe present disclosure.

Certain normal physiological processes are also associated withangiogenesis, for example, ovulation, menstruation, and placentation.The angiogenesis inhibiting proteins of the present disclosure areuseful in the treatment of disease of excessive or abnormal stimulationof endothelial cells. These diseases include, but are not limited to,intestinal adhesions, atherosclerosis, scleroderma, and hypertrophicscars, i.e., keloids. They are also useful in the treatment of diseasesthat have angiogenesis as a pathologic consequence such as cat scratchdisease (Rochele minalia quintosa) and ulcers (Helicobacter pylori).

General angiogenesis-inhibiting proteins can be used as birth controlagents by reducing or preventing uterine vascularization required forembryo implantation. Thus, the present disclosure provides an effectivebirth control method when an amount of the inhibitory protein sufficientto prevent embryo implantation is administered to a female. In oneaspect of the birth control method, an amount of the inhibiting proteinsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method. While notwanting to be bound by this statement, it is believed that inhibition ofvascularization of the uterine endometrium interferes with implantationof the blastocyst. Similar inhibition of vascularization of the mucosaof the uterine tube interferes with implantation of the blastocyst,preventing occurrence of a tubal pregnancy. Administration methods mayinclude, but are not limited to, pills, injections (intravenous,subcutaneous, intramuscular), suppositories, vaginal sponges, vaginaltampons, and intrauterine devices. It is also believed thatadministration of angiogenesis inhibiting agents of the presentdisclosure will interfere with normal enhanced vascularization of theplacenta, and also with the development of vessels within a successfullyimplanted blastocyst and developing embryo and fetus.

In the eye, angiogenesis is associated with, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, and retrolental fibroplasias. Thetherapeutic agents disclosed herein may be administered intra-ocularlyor by other local administration to the eye. Other diseases associatedwith angiogenesis in the eye include, but are not limited to, epidemickeratoconjunctivitis, vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, herpes simplex infections, herpes zoster infections, protozoaninfections, Kaposi sarcoma, Mooren ulcer, Terrien's marginaldegeneration, mariginal keratolysis, rheumatoid arthritis, systemiclupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven'sJohnson disease, periphigoid radial keratotomy, corneal graft rejection,sickle cell anemia, sarcoid, pseudoxanthoma elasticum, Pagets disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme disease, systemic lupuserythematosis, retinopathy of prematurity, Eales disease, Bechetsdisease, infections causing a retinitis or choroiditis, presumed ocularhistoplasmosis, Bests disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications. Other diseasesinclude, but are not limited to, diseases associated with rubeosis(neovasculariation of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy.

Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of a therapeutic agent, or by insertionof a sustained release device that releases a therapeutic agent. Atherapeutic agent may be delivered in a pharmaceutically acceptableophthalmic vehicle, such that the compound is maintained in contact withthe ocular surface for a sufficient time period to allow the compound topenetrate the corneal and internal regions of the eye, as for examplethe anterior chamber, posterior chamber, vitreous body, aqueous humor,vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera.The pharmaceutically-acceptable ophthalmic vehicle may, for example, bean ointment, vegetable oil or an encapsulating material. Alternatively,the therapeutic agents of the disclosure may be injected directly intothe vitreous and aqueous humour. In a further alternative, the compoundsmay be administered systemically, such as by intravenous infusion orinjection, for treatment of the eye.

One or more therapeutic agents can be administered. The methods of thedisclosure also include co-administration with other medicaments thatare used to treat conditions of the eye. When administering more thanone agent or a combination of agents and medicaments, administration canoccur simultaneously or sequentially in time. The therapeutic agentsand/or medicaments may be administered by different routes ofadministration or by the same route of administration. In oneembodiment, a therapeutic agent and a medicament are administeredtogether in an ophthalmic pharmaceutical formulation.

In one embodiment, a therapeutic agent is used to treat a diseaseassociated with angiogenesis in the eye by concurrent administrationwith other medicaments that act to block angiogenesis by pharmacologicalmechanisms. Medicaments that can be concurrently administered with atherapeutic agent of the disclosure include, but are not limited to,pegaptanib (Macugen™), ranibizumab (Lucentis™), squalamine lactate(Evizon™ heparinase, and glucocorticoids (e.g. Triamcinolone). In oneembodiment, a method is provided to treat a disease associated withangiogenesis is treated by administering an ophthalmic pharmaceuticalformulation containing at least one therapeutic agent disclosed hereinand at least one of the following medicaments: pegaptanib (Macugen™),ranibizumab (Lucentis™), squalamine lactate (Evizon™), heparinase, andglucocorticoids (e.g. Triamcinolone).

In other embodiments, ENG polypeptides can be used to treat a patientwho suffers from a cardiovascular disorder or condition associated withBMP-9 or BMP-10 but not necessarily accompanied by angiogenesis.Exemplary disorders of this kind include, but are not limited to, heartdisease (including myocardial disease, myocardial infarct, anginapectoris, and heart valve disease); renal disease (including chronicglomerular inflammation, diabetic renal failure, and lupus-related renalinflammation); disorders of blood pressure (including systemic andpulmonary types); disorders associated with atherosclerosis or othertypes of arteriosclerosis (including stroke, cerebral hemorrhage,subarachnoid hemorrhage, angina pectoris, and renal arteriosclerosis);thrombotic disorders (including cerebral thrombosis, pulmonarythrombosis, thrombotic intestinal necrosis); complications of diabetes(including diabetes-related retinal disease, cataracts, diabetes-relatedrenal disease, diabetes-related neuropathology, diabetes-relatedgangrene, and diabetes-related chronic infection); vascular inflammatorydisorders (systemic lupus erythematosus, joint rheumatism, jointarterial inflammation, large-cell arterial inflammation, Kawasakidisease, Takayasu arteritis, Churg-Strauss syndrome, andHenoch-Schoenlein pupura); and cardiac disorders such as congenitalheart disease, cardiomyopathy (e.g., dilated, hypertrophic, restrictivecardiomyopathy), and congestive heart failure. The ENG polypeptide canbe administered to the subject alone, or in combination with one or moreagents or therapeutic modalities, e.g., therapeutic agents, which areuseful for treating BMP-9/10 associated cardiovascular disorders and/orconditions. In one embodiment, the second agent or therapeutic modalityis chosen from one or more of: angioplasty, beta blockers,anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators,hormone antagonists, endothelin antagonists, calcium channel blockers,phosphodiesterase inhibitors, angiotensin type 2 antagonists and/orcytokine blockers/inhibitors.

In still other embodiments, ENG polypeptides may be useful in thetreatment or prevention of fibrosis. As used herein, the term “fibrosis”refers to the aberrant formation or development of excess fibrousconnective tissue by cells in an organ or tissue. Although processesrelated to fibrosis can occur as part of normal tissue formation orrepair, dysregulation of these processes can lead to altered cellularcomposition and excess connective tissue deposition that progressivelyimpairs to tissue or organ function. The formation of fibrous tissue canresult from a reparative or reactive process. Fibrotic disorders orconditions include, but are not limited to, fibroproliferative disordersassociated with vascular diseases, such as cardiac disease, cerebraldisease, and peripheral vascular disease, as well as tissues and organsystems including the heart, skin, kidney, lung, peritoneum, gut, andliver (as disclosed in, e.g., Wynn, 2004, Nat Rev 4:583-594,incorporated herein by reference). Exemplary disorders that can betreated include, but are not limited to, renal fibrosis, includingnephropathies associated with injury/fibrosis, e.g., chronicnephropathies associated with diabetes (e.g., diabetic nephropathy),lupus, scleroderma, glomerular nephritis, focal segmental glomerularsclerosis, and IgA nephropathy; lung or pulmonary fibrosis, e.g.,idiopathic pulmonary fibrosis, radiation induced fibrosis, chronicobstructive pulmonary disease (COPD), scleroderma, and chronic asthma;gut fibrosis, e.g., scleroderma, and radiation-induced gut fibrosis;liver fibrosis, e.g., cirrhosis, alcohol-induced liver fibrosis, biliaryduct injury, primary biliary cirrhosis, infection or viral induced liverfibrosis, congenital hepatic fibrosis and autoimmune hepatitis; andother fibrotic conditions, such as cystic fibrosis, endomyocardialfibrosis, mediastinal fibrosis, pleural fibrosis, sarcoidosis,scleroderma, spinal cord injury/fibrosis, myelofibrosis, vascularrestenosis, atherosclerosis, cystic fibrosis of the pancreas and lungs,injection fibrosis (which can occur as a complication of intramuscularinjections, especially m children), endomyocardial fibrosis, idiopathicpulmonary fibrosis of the lung, mediastinal fibrosis, mylcofibrosis,retroperitoneal fibrosis, progressive massive fibrosis, a complicationof coal workers' pneumoconiosis, and nephrogenic systemic fibrosis.

As used herein, the terms “fibrotic disorder”, “fibrotic condition,” and“fibrotic disease,” are used interchangeably to refer to a disorder,condition or disease characterized by fibrosis. Examples of fibroticdisorders include, but are not limited to vascular fibrosis, pulmonaryfibrosis (e.g., idiopathic pulmonary fibrosis), pancreatic fibrosis,liver fibrosis (e.g., cirrhosis), renal fibrosis, musculoskeletalfibrosis, cardiac fibrosis (e.g., endomyocardial fibrosis, idiopathicmyocardiopathy), skin fibrosis (e.g., scleroderma, post-traumatic,operative cutaneous scarring, keloids and cutaneous keloid formation),eye fibrosis (e.g., glaucoma, sclerosis of the eyes, conjunctival andcorneal scarring, and pterygium), progressive systemic sclerosis (PSS),chronic graft-versus-host disease, Peyronie's disease, post-cystoscopicurethral stenosis, idiopathic and pharmacologically inducedretroperitoneal fibrosis, mediastinal fibrosis, progressive massivefibrosis, proliferative fibrosis, and neoplastic fibrosis.

As used herein, the term “cell” refers to any cell prone to undergoing afibrotic response, including, but not limited to, individual cells,tissues, and cells within tissues and organs. The term cell, as usedherein, includes the cell itself, as well as the extracellular matrix(ECM) surrounding a cell. For example, inhibition of the fibroticresponse of a cell, includes, but is not limited to the inhibition ofthe fibrotic response of one or more cells within the lung (or lungtissue); one or more cells within the liver (or liver tissue); one ormore cells within the kidney (or renal tissue); one or more cells withinmuscle tissue; one or more cells within the heart (or cardiac tissue);one or more cells within the pancreas; one or more cells within theskin; one or more cells within the bone, one or more cells within thevasculature, one or more stem cells, or one or more cells within theeye.

The methods and compositions of the present invention can be used totreat and/or prevent fibrotic disorders. Exemplary types of fibroticdisorders include, but are not limited to, vascular fibrosis, pulmonaryfibrosis (e.g., idiopathic pulmonary fibrosis), pancreatic fibrosis,liver fibrosis (e.g., cirrhosis), renal fibrosis, musculoskeletalfibrosis, cardiac fibrosis (e.g., endomyocardial fibrosis, idiopathicmyocardiopathy), skin fibrosis (e.g., scleroderma, post-traumatic,operative cutaneous scarring, keloids and cutaneous keloid formation),eye fibrosis (e.g., glaucoma, sclerosis of the eyes, conjunctival andcorneal scarring, and pterygium), progressive systemic sclerosis (PSS),chronic graft vcrsus-host disease, Peyronie's disease, post-cystoscopicurethral stenosis, idiopathic and pharmacologically inducedretroperitoneal fibrosis, mediastinal fibrosis, progressive massivefibrosis, proliferative fibrosis, neoplastic fibrosis, Dupuytren'sdisease, strictures, and radiation induced fibrosis. In a particularembodiment, the fibrotic disorder is not myelofibrosis.

The present invention contemplates the use of ENG polypeptides incombination with one or more other therapeutic modalities. Thus, inaddition to the use of ENG polypeptides, one may also administer to thesubject one or more “standard” therapies for treating fibroticdisorders. For example, the ENG polypeptides can be administered incombination with (i.e., together with) cytotoxins, immunosuppressiveagents, radiotoxic agents, and/or therapeutic antibodies. Particularco-therapeutics contemplated by the present invention include, but arenot limited to, steroids (e.g., corticosteroids, such as Prednisone),immune-suppressing and/or anti-inflammatory agents (e.g.,gamma-interferon, cyclophosphamide, azathioprine, methotrexate,penicillamine, cyclosporine, colchicines, antithymocyte globulin,mycophenolate mofetil, and hydroxychloroquine), cytotoxic drugs, calciumchannel blockers (e.g., nifedipine), angiotensin converting enzymeinhibitors (ACE) inhibitors, para-aminobenzoic acid (PABA), dimethylsulfoxide, transforming growth factor-beta (TGF-β) inhibitors,interleukin-5 (IL-5) inhibitors, and pan caspase inhibitors.

Additional anti-fibrotic agents that may be used in combination with ENGpolypeptides include, but are not limited to, lectins (as described in,for example, U.S. Pat. No. 7,026,283, the entire contents of which isincorporated herein by reference), as well as the anti-fibrotic agentsdescribed by Wynn et al (2007, J Clin Invest 117:524-529, the entirecontents of which is incorporated herein by reference). For example,additional anti-fibrotic agents and therapies include, but are notlimited to, various anti-inflammatory/immunosuppressive/cytotoxic drugs(including colchicine, azathioprine, cyclophosphamide, prednisone,thalidomide, pentoxifylline and theophylline), TGF-0 signaling modifiers(including relaxin, SMAD7, HGF, and BMP7, as well as TGF-131, TGFβRI,TGFβRII, EGR-I, and CTGF inhibitors), cytokine and cytokine receptorantagonists (inhibitors of IL-1β, IL-5, IL-6, IL-13, IL-21, IL-4R,IL-13Rα1, GM-CSF, TNF-α, oncostatin M, W1SP-I, and PDGFs), cytokines andchemokines (IFN-γ, IFN-α/β, IL-12, IL-10, HGF, CXCL10, and CXCL11),chemokine antagonists (inhibitors of CXCL1, CXCL2, CXCL12, CCL2, CCL3,CCL6, CCL17, and CCL18), chemokine receptor antagonists (inhibitors ofCCR2, CCR3, CCRS, CCR7, CXCR2, and CXCR4), TLR antagonists (inhibitorsof TLR3, TLR4, and TLR9), angiogenesis antagonists (VEGF-specificantibodies and adenosine deaminase replacement therapy),antihypertensive drugs (beta blockers and inhibitors of ANG 11, ACE, andaldosterone), vasoactive substances (ET-1 receptor antagonists andbosetan), inhibitors of the enzymes that synthesize and process collagen(inhibitors of prolyl hydroxylase), B cell antagonists (rituximab),integrin/adhesion molecule antagonists (molecules that block α1β1 andαvβ6 integrins, as well as inhibitors of integrin-linked kinase, andantibodies specific for ICAM-I and VCAM-I), proapoptotic drugs thattarget myofibroblasts, MMP inhibitors (inhibitors of MMP2, MMP9, andMMP12), and T1MP inhibitors (antibodies specific for TIMP-1).

The ENG polypeptide and the co-therapeutic agent or co-therapy can beadministered in the same formulation or separately. In the case ofseparate administration, the ENG polypeptide can be administered before,after, or concurrently with the co-therapeutic or co-therapy. One agentmay precede or follow administration of the other agent by intervalsranging from minutes to weeks. In embodiments where two or moredifferent kinds of therapeutic agents are applied separately to asubject, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that thesedifferent kinds of agents would still be able to exert an advantageouslycombined effect on the target tissues or cells.

In still other embodiments, ENG polypeptides may be useful in thetreatment of inflammatory disorders or conditions likely to beBMP9-related but not already noted above. Exemplary disorders includeliver disease (including acute hepatitis, chronic hepatitis, andcirrhosis); thoracic or abdominal edema; chronic pancreatic disease;allergies (including nasal allergy, asthma, bronchitis, and atopicdermatitis); Alzheimer's disease; Raynaud's syndrome; and diffusesclerosis.

6. Formulations and Effective Doses

The therapeutic agents described herein may be formulated intopharmaceutical compositions. Pharmaceutical compositions for use inaccordance with the present disclosure may be formulated in conventionalmanner using one or more physiologically acceptable carriers orexcipients. Such formulations will generally be substantially pyrogenfree, in compliance with most regulatory requirements.

In certain embodiments, the therapeutic method of the disclosureincludes administering the composition systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this disclosure is in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the ENG signalingantagonists which may also optionally be included in the composition asdescribed above, may be administered simultaneously or sequentially withthe subject compounds (e.g., ENG polypeptides) in the methods disclosedherein.

Typically, protein therapeutic agents disclosed herein will beadministered parentally, and particularly intravenously orsubcutaneously. Pharmaceutical compositions suitable for parenteraladministration may comprise one or more ENG polypeptides in combinationwith one or more pharmaceutically acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In one embodiment, the ENG polypeptides disclosed herein areadministered in an ophthalmic pharmaceutical formulation. In someembodiments, the ophthalmic pharmaceutical formulation is a sterileaqueous solution, preferable of suitable concentration for injection, ora salve or ointment. Such salves or ointments typically comprise one ormore ENG polypeptides disclosed herein dissolved or suspended in asterile pharmaceutically acceptable salve or ointment base, such as amineral oil-white petrolatum base. In salve or ointment compositions,anhydrous lanolin may also be included in the formulation. Thimerosal orchlorobutanol are also preferably added to such ointment compositions asantimicrobial agents. In one embodiment, the sterile aqueous solution isas described in U.S. Pat. No. 6,071,958.

The disclosure provides formulations that may be varied to include acidsand bases to adjust the pH; and buffering agents to keep the pH within anarrow range. Additional medicaments may be added to the formulation.These include, but are not limited to, pegaptanib, heparinase,ranibizumab, or glucocorticoids. The ophthalmic pharmaceuticalformulation according to the disclosure is prepared by asepticmanipulation, or sterilization is performed at a suitable stage ofpreparation.

The compositions and formulations may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Expression of Fusion Protein Comprising Full-LengthExtracellular Domain of Human ENG

Applicants constructed a soluble endoglin (ENG) fusion protein(hENG(26-586)-hFc) in which the full-length extracellular domain (ECD)of human ENG (FIG. 9, SEQ ID NO: 9) was attached to a human IgG₁ Fcdomain (FIG. 11, SEQ ID NO: 11) with a minimal linker between thesedomains. hENG(26-586)-hFc was expressed by transient transfection in HEK293 cells. In brief, HEK 293 cells were set up in a 500-ml spinner at6×10⁵ cells/ml in a 250 ml volume of Freestyle media (Invitrogen) andgrown overnight. Next day, these cells were treated with DNA:PEI (1:1)complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml mediawas added and cells were grown for 7 days. Conditioned media washarvested by spinning down the cells and concentrated. For expression inCHO cells, ENG polypeptide constructs were transfected into a CHO DUKX B11 cell line. Clones were selected in methotrexate (MTX), typically atan initial concentration of 5 nM or 10 nM, and optionally followed byamplification in 50 nM MTX to increase expression. A high expressingclone could be identified by dilution cloning and adapted to serum-freesuspension growth to generate conditioned media for purification.Optionally, a ubiquitous chromatin opening element (UCOE) may beincluded in the vector to facilitate expression. See, e.g.,Cytotechnology. 2002 January; 38(1-3):43-6.

Three different leader sequences may be used:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 13) MKFLVNVALVFMVVYISYIYA(ii) Tissue plasminogen activator (TPA): (SEQ ID NO: 14)MDAMKRGLCCVLLLCGAVFVSP (iii) Native human ENG: (SEQ ID NO: 15)MDRGTLPLAVALLLASCSLSPTSLA

The selected form of hENG(26-586)-hFc uses the TPA leader, has theunprocessed amino acid sequence shown in FIG. 13 (SEQ ID NO: 16), and isencoded by the nucleotide sequence shown in FIG. 14 (SEQ ID NO: 17).Applicants also envision an alternative hENG(26-586)-hFc sequence withTPA leader (FIG. 15, SEQ ID NO: 18) comprising an N-terminally truncatedhFc domain (FIG. 12, SEQ ID NO: 12) attached to hENG(26-586) by a TGGGlinker. Purification was achieved using a variety of techniques,including, for example, filtration of conditioned media, followed byprotein A chromatography, elution with low-pH (3.0) glycine buffer,sample neutralization, and dialysis against PBS. Purity of samples wasevaluated by analytical size-exclusion chromatography, SDS-PAGE, silverstaining, and Western blot. Analysis of mature protein confirmed theexpected N-terminal sequence.

Example 2 Expression of Fusion Protein Comprising Full-LengthExtracellular Domain of Murine ENG

Applicants constructed a soluble murine ENG fusion protein(mENG(27-581)-mFc) in which the full-length extracellular domain ofmurine ENG (FIG. 10, SEQ ID NO: 10) was fused to a murine IgG_(2a) Fcdomain with a minimal linkers between these domains. mENG(27-581)-mFcwas expressed by transient transfection in HEK 293 cells.

The selected form of mENG(27-581)-mFc uses the TPA leader, has theunprocessed amino acid sequence shown in FIG. 16 (SEQ ID NO: 19), and isencoded by the nucleotide sequence shown in FIG. 17 (SEQ ID NO: 20).Purification was achieved by filtration of conditioned media fromtransfected HEK 293 cells, followed by protein A chromatography. Purityof samples was evaluated by analytical size-exclusion chromatography,SDS-PAGE, silver staining, and Western blot analysis.

Example 3 Selective Binding of BMP-9/BMP-10 to Proteins ComprisingFull-Length Extracellular ENG Domain

Considered a co-receptor, ENG is widely thought to function byfacilitating the binding of TGF-β1 and -3 to multiprotein complexes oftype I and type II receptors. To investigate the possibility of directligand binding by isolated ENG, Applicants used surface plasmonresonance (SPR) methodology (Biacore™ instrument) to screen for bindingof captured proteins comprising the full-length extracellular domain ofENG to a variety of soluble human TGF-β family ligands.

Construct Binding hENG(26-586)- mENG(27-581)- Ligand hFc* hENG(26-586)**hFc*** hBMP-2 − − − hBMP-2/7 − − − hBMP-7 − − − hBMP-9 ++++ ++++ ++++hBMP-10 ++++ ++++ ++++ hTGF-β1 − − − hTGF-β2 − − − hTGF-β3 − − −hActivin A − − − *[hBMP-9], [hBMP-10] = 2.5 nM; all other ligands testedat 100 nM **[hBMP-9], [hBMP-10] = 2.5 nM; all other ligands tested at 25nM ***[hBMP-9], [hBMP-10] = 0.5 nM; [hTGF-β1], [hTGF-β2], [hTGF-β3] = 10nM; all other ligands tested at 25 nM

As shown in this table, binding affinity to hENG(26-586)-hFc was high(++++, K_(D)<1 nM) for hBMP-9 and hBMP-10 as evaluated at low ligandconcentrations. Even at concentrations 40-fold higher, binding ofTGF-β1, TGF-β2, TGF-β3, activin A, BMP-2, and BMP-7 to hENG(26-586)-hFcwas undetectable (−). For this latter group of ligands, lack of directbinding to isolated ENG fusion protein is noteworthy becausemultiprotein complexes of type I and type II receptors have been shownto bind most of them better in the presence of ENG than in its absence.As also shown in the table above, similar results were obtained whenligands were screened for their ability to bind immobilized hENG(26-586)(R&D Systems, catalog #1097-EN), a human variant with no Fc domain, ortheir ability to bind captured mENG(27-581)-hFc (R&D Systems, catalog#1320-EN), consisting of the extracellular domain of murine ENG(residues 27-581) attached to the Fc domain of human IgG₁ via asix-residue linker sequence (IEGRMD). Characterization by SPR (FIGS. 18,19) determined that captured hENG(26-586)-hFc binds soluble BMP-9 with aK_(D) of 29 pM and soluble BMP-10 with a K_(D) of 400 pM. Thus,selective high-affinity binding of BMP-9 and BMP-10 is a previouslyunrecognized property of the ENG extracellular domain that isgeneralizable across species.

Example 4 Soluble Extracellular Domain of hENG Inhibits Binding ofBMP-9/BMP-10 to ALK1 and Other Cognate Receptors

BMP-9 and BMP-10 are high-affinity ligands at the type I receptor ALK1(activin receptor-like kinase 1). An SPR-based assay was used todetermine the effect of soluble hENG(26-586) (R&D Systems, catalog#1097-EN) on binding of BMP-9 and BMP-10 to ALK1. ALK1-hFc was capturedand then exposed to solutions containing soluble hENG(26-586) premixedwith BMP-9 in various ratios. As shown in FIG. 20, soluble hENG(26-586)inhibited binding of BMP-9 to ALK1-Fc in a concentration-dependentmanner with an IC₅₀ less than 10 nM. Similar results were obtained withBMP-10 (FIG. 21). Separate experiments have demonstrated that solublehENG(26-586) does not bind ALK1 and therefore does not inhibit ligandbinding to ALK1 by this mechanism. Indeed, additional SPR-basedexperiments indicate that soluble hENG(26-586) binds neither type Ireceptors ALK2-ALK7 nor type II receptors such as activin receptor IIA,activin receptor IIB, bone morphogenetic protein receptor II, and TGF-βreceptor II. These results provide further evidence that ENG inhibitsbinding of BMP-9 and BMP-10 to ALK1 primarily through a directinteraction with these ligands.

Taken together, these data demonstrate that soluble ENG-Fc chimericproteins as well as non-chimeric soluble ENG can be used as antagonistsof BMP-9 and BMP-10 signaling through multiple signaling pathways,including ALK1.

Example 5 Effect of mENG(27-581)-hFc on Human Umbilical Vein EndothelialCells (HUVEC) in Culture

Applicants investigated the angiogenic effect of mENG(27-581)-hFc in aHUVEC-based culture system. HUVECs were cultured on a polymerizedMatrigel substrate, and the effect of test articles on formation ofendothelial-cell tubes (cords) was assessed by phase-contrast microscopyafter 12 h exposure. Cords possessing single-cell width and at leastthree branches were identified visually, and computer-assisted imageanalysis was used to determine the total length of such cords. Meanvalues are based on duplicate culture wells per experimental condition,with each well characterized as the average of three fields ofobservation. Compared to basal conditions (no treatment), the stronginducing agent endothelial cell growth substance (ECGS, 0.2 μg/ml)doubled mean cord length (FIG. 22). mENG(27-581)-hFc (R&D Systems,catalog #1320-EN; 10 μg/ml) cut this increase by nearly 60%, an effectspecific for stimulated conditions because the same concentration ofmENG(27-581)-hFc had little effect in the absence of ECGS (FIG. 22).These results demonstrate that ENG-Fc fusion protein can inhibitendothelial cell aggregation under otherwise stimulated conditions in acell-culture model of angiogenesis.

Example 6 ENG-Fc Inhibits VEGF-Inducible Angiogenesis in a ChickChorioallantoic Membrane (CAM) Assay

A chick chorioallantoic membrane (CAM) assay system was used toinvestigate effects of ENG-Fc fusion protein on angiogenesis. In brief,nine-day-old fertilized chick embryos were maintained in an eggincubator at controlled temperature (37° C.) and humidity (60%). The eggshell was softened with alcohol, punctured with a tiny hole to create a“blister” between the shell membrane and CAM, and removed to create awindow overlying prominent blood vessels. Small filter disks weretreated with VEGF (50 ng daily) in the presence or absence ofmENG(27-581)-hFc protein (R&D Systems, catalog #1320-EN; 14 μg daily)dissolved in buffer (pH 7.4) containing 0.01 M HEPES, 0.5 M NaCl, 3 mMEDTA, 0.005% v/v Surfactant P20, and 0.5 mg/ml bovine serum albumin.Filter disks containing test article were then inserted through theopening and apposed to the CAM. Eggs (n=8 per group) were treated withfresh test article daily for three days, and on the fourth day thenumber of blood vessels associated with the filter disk was determinedby visual inspection with the assistance of an egg lamp.

As expected, VEGF treatment in the CAM assay system increased the numberof blood vessels markedly over that of vehicle. The number of additionalblood vessels induced by VEGF treatment was decreased by 65% withconcurrent mENG(27-581)-hFc treatment (FIG. 23). SPR-based studiesindicate that VEGF does not bind mENG(27-581)-mFc, and thus effects ofmENG(27-581)-hFc on angiogenesis in the present CAM experiment were notdue to a direct interaction between the fusion protein and VEGF. Theforegoing results indicate that ENG-Fc can significantly inhibit thewell-established angiogenic effect of VEGF in an in vivo model withoutcontacting VEGF itself.

Example 7 Effect of mENG(27-581)-mFc on Angiogenesis in a MouseAngioreactor Assay

Effects of ENG-Fc fusion protein on angiogenesis were furtherinvestigated in a mouse angioreactor assay, also known as a directed invivo angiogenesis assay (DIVAA™; Guedez et al., 2003, Am J Pathol162:1431-1439), which was performed according to instructions of themanufacturer (Trevigen®). In brief, hollow cylinders made ofimplant-grade silicone and closed at one end were filled with 20 μl ofbasement membrane extract (BME) premixed with or without a combinationof basic fibroblast growth factor (FGF-2, 1.8 g) and VEGF (600 ng).After the BME had gelled, angioreactors were implanted subcutaneously inathymic nude mice (four per mouse). Mice were treated daily withmENG(27-581)-mFc (10 mg/kg, s.c.) or vehicle (Tris-buffered saline) for11 days, at which time mice were injected with fluoresceinisothiocyanate (FITC)-labeled dextran (20 mg/kg, i.v.) and euthanized 20min later. Angioreactors were removed, and the amount of FITC-dextrancontained in each was quantified with a fluorescence plate reader(Infinite® M200, Tecan) at 485 nm excitation/520 nm emission as an indexof blood vessel formation. As shown in FIG. 24, addition of FGF-2 andVEGF to the BME led to a significant increase in vascularization withinthe angioreactors at study completion, whereas the concurrentadministration of mENG(27-581)-mFc prevented this increase completely.These results obtained in a mammalian system complement those obtainedwith the CAM assay described above and demonstrate the in vivoanti-angiogenic activity of ENG-Fc fusion proteins incorporating afull-length ENG extracellular domain.

Example 8 Expression of Variants with Truncated hENG ExtracellularDomain

Applicants generated soluble ENG fusion proteins in which truncatedvariants of the human ENG ECD were fused to a human IgG₁ Fc domain witha minimal linker. These variants are listed below, and the structures ofselected variants are shown schematically in FIG. 25.

Stable Transient Expression Human Construct Expression Purified (CHOCells) Full Length hENG(26-586)-hFc HEK 293 Yes Yes Carboxy-hENG(26-581)-hFc HEK 293 Yes No Terminal hENG(26-437)-hFc HEK 293 Yes NoTruncations hENG(26-378)-hFc HEK 293 Yes No hENG(26-359)-hFc HEK 293 YesYes hENG(26-346)-hFc HEK 293 Yes Yes hENG(26-332)-hFc HEK 293 Yes NohENG(26-329)-hFc HEK 293 Yes No hENG(26-257)-hFc HEK 293 Yes No Amino-hENG(360-586)-hFc HEK 293 Yes No Terminal hENG(438-586)-hFc HEK 293 YesNo Truncations hENG(458-586)-hFc COS No No Double hENG(61-346)-hFc HEK293 Yes No Truncations hENG(129-346)-hFc HEK 293 Yes NohENG(133-346)-hFc HEK 293 Yes No hENG(166-346)-hFc HEK 293 Yes NohENG(258-346)-hFc HEK 293 Yes No hENG(360-581)-hFc HEK 293 Yes NohENG(360-457)-hFc COS No No hENG(360-437)-hFc COS No NohENG(458-581)-hFc COS No NoThese variants were expressed by transient transfection in HEK 293 cellsor COS cells, as indicated.

The selected form of hENG(26-437)-hFc uses the TPA leader, has theunprocessed amino acid sequence shown in FIG. 26 (SEQ ID NO: 21), and isencoded by the nucleotide sequence shown in FIG. 27 (SEQ ID NO: 22). Theselected form of hENG(26-378)-hFc also uses the TPA leader, has theunprocessed amino acid sequence shown in FIG. 28 (SEQ ID NO: 23), and isencoded by the nucleotide sequence shown in FIG. 29 (SEQ ID NO: 24). Theselected form of hENG(26-359)-hFc also uses the TPA leader, has theunprocessed amino acid sequence shown in FIG. 30 (SEQ ID NO: 25), and isencoded by the nucleotide sequence shown in FIG. 31 (SEQ ID NO: 26).Applicants also envision an alternative hENG(26-359)-hFc sequence withTPA leader (FIG. 32, SEQ ID NO: 27) comprising an N-terminally truncatedhFc domain (FIG. 12, SEQ ID NO: 12) attached to hENG(26-359) by a TGGGlinker. The nucleotide sequence encoding this alternativehENG(26-359)-hFc protein is shown in FIG. 33 (SEQ ID NO: 28). Theselected form of hENG(26-346)-hFc uses the TPA leader, has theunprocessed amino acid sequence shown in FIG. 34 (SEQ ID NO: 29)comprising an N-terminally truncated hFc domain, and is encoded by thenucleotide sequence shown in FIG. 35 (SEQ ID NO: 30).

Selected hENG-hFc variants, each with an N-terminally truncated Fcdomain (SEQ ID NO: 12), were stably expressed in CHO cells (usingmethodology described above) and purified from conditioned media byfiltration and protein A chromatography. Analysis of mature proteinexpressed in CHO cells confirmed the N-terminal sequences ofhENG(26-359)-hFc and hENG(26-346)-hFc to be as expected. On the basis ofprotein yield (uncorrected for differences in theoretical molecularweight), hENG(26-346)-hFc (90 mg/liter) was superior to bothhENG(26-359)-hFc (9 mg/liter) and full-length hENG(26-586)-hFc (31mg/liter). As shown in FIG. 36, analysis of these purified samples bysize-exclusion chromatography revealed the quality of hENG(26-346)-hFcprotein (96% monomeric) to be superior to that of hENG(26-359)-hFcprotein (84% monomeric) and equivalent to that of hENG(26-586)-hFcprotein (96% monomeric). Thus, greater levels of high-molecular-weightaggregates require the use of additional purification steps forhENG(26-359)-hFc compared to hENG(26-346)-hFc.

Example 9 High-Affinity Binding of BMP-9/BMP-10 to Truncated hENG-hFcVariants

Applicants used SPR methodology to screen the following hENG-hFc proteinvariants for high-affinity binding to human BMP-9 and BMP-10. In theseexperiments, captured hENG-hFc proteins were exposed to soluble BMP-9 orBMP-10 at 100 nM each.

Binding to hBMP-9 Human Construct and hBMP-10 Full LengthhENG(26-586)-hFc ++++ Carboxy-Terminal hENG(26-581)-hFc ++++ TruncationshENG(26-437)-hFc ++++ hENG(26-378)-hFc ++++ hENG(26-359)-hFc ++++hENG(26-346)-hFc ++++ hENG(26-332)-hFc − hENG(26-329)-hFc −hENG(26-257)-hFc − Amino-Terminal hENG(360-586)-hFc − TruncationshENG(438-586)-hFc − hENG(458-586)-hFc − Double TruncationshENG(61-346)-hFc − hENG(129-346)-hFc − hENG(133-346)-hFc −hENG(166-346)-hFc − hENG(258-346)-hFc − hENG(360-581)-hFc −hENG(360-457)-hFc − hENG(360-437)-hFc − hENG(458-581)-hFc − ++++ KD <1nM − Binding undetectable

As indicated in the table above, high-affinity binding to BMP-9 andBMP-10 was observed only for the full-length construct and forC-terminally truncated variants as short as hENG(26-346)-hFc.High-affinity binding to BMP-9 and BMP-10 was lost for all N-terminaltruncations of greater than 61 amino acids that were tested.

A panel of ligands were screened for potential binding to the C-terminaltruncated variants hENG(26-346)-hFc, hENG(26-359)-hFc, andhENG(26-437)-hFc. High-affinity binding of these three proteins wasselective for BMP-9 and BMP-10. Neither hENG(26-346)-hFc,hENG(26-359)-hFc, nor hENG(26-437)-hFc displayed detectable binding toBMP-2, BMP-7, TGF-β1, TGF-β2, TGF-β3, or activin A, even at high ligandconcentrations.

Construct Binding hENG(26-346)- hENG(26-359)- hENG(26-437)- Ligand hFc*hFc** hFc** hBMP-2 − − − hBMP-2/7 − − − hBMP-7 − − − hBMP-9 ++++ ++++++++ hBMP-10 ++++ ++++ ++++ hTGF-β1 − − − hTGF-β2 − − − hTGF-β3 − − −hActivin A − − − *[hBMP-9], [hBMP-10] = 5 nM; [hTGF-β3] = 50 nM; allother ligands tested at 100 nM **[hBMP-9], [hBMP-10] = 5 nM; [hTGF-β3] =50 nM; all other ligands tested at 100 nM ++++ KD <1 nM − Bindingundetectable

Applicants used SPR methodology to compare the kinetics of BMP-9 bindingby five constructs: hENG(26-586)-hFc, hENG(26-437)-hFc,hENG(26-378)-hFc, hENG(26-359)-hFc, and hENG(26-346)-hFc. FIG. 37 showsbinding curves for several of the constructs, and the table below listscalculated values for the equilibrium dissociation constants anddissociation rate constants (k_(d)). The affinity of human BMP-9 forhENG(26-359)-hFc or hENG(26-346)-hFc (with K_(D)s in the low picomolarrange) was nearly an order of magnitude stronger than for thefull-length construct. It is highly desirable for ligand traps such asENG-Fc to exhibit a relatively slow rate of ligand dissociation, so theten-fold improvement (decrease) in the BMP-9 dissociation rate forhENG(26-346)-hFc compared to the full-length construct is particularlynoteworthy.

Ligand Construct K_(D) (×10⁻¹²M) k_(d) (×10⁻⁴ s⁻¹) hBMP-9hENG(26-586)-hFc* 33 25 hENG(26-437)-hFc** 19 14 hENG(26-378)-hFc** 6.73.4 hENG(26-359)-hFc* 4.2 3.5 hENG(26-346)-hFc* 4.3 2.4*CHO-cell-derived protein **HEK293-cell-derived protein

As shown below, each of the truncated variants also bound BMP-10 withhigher affinity, and with better kinetics, compared to the full-lengthconstruct. Even so, the truncated variants differed in their degree ofpreference for BMP-9 over BMP-10 (based on K_(D) ratio), withhENG(26-346)-hFc displaying the largest differential andhENG(26-437)-hFC the smallest. This difference in degree of ligandpreference among the truncated variants could potentially translate intomeaningful differences in their activity in vivo.

Ligand Construct K_(D) (×10⁻¹²M) k_(d) (×10⁻⁴ s⁻¹) hBMP-10hENG(26-586)-hFc* 490 110 hENG(26-437)-hFc** 130 28 hENG(26-378)-hFc**95 19 hENG(26-359)-hFc* 86 23 hENG(26-346)-hFc* 140 28 *CHO-cell-derivedprotein **HEK293-cell-derived protein

The foregoing results indicate that fusion proteins comprising certainC-terminally truncated variants of the hENG ECD display high-affinitybinding to BMP-9 and BMP-10 but not to a variety of other TGF-β familyligands, including TGF-β1 and TGF-β3. In particular, the truncatedvariants hENG(26-359)-hFc, hENG(26-346)-hFc, and hENG(26-378)-hFcdisplay higher binding affinity at equilibrium and improved kineticproperties for BMP-9 compared to both the full-length constructhENG(26-586)-hFc and the truncated variant hENG(26-437)-hFc.

Example 10 Prediction of Secondary Structure for ENG N-Terminal Region

As disclosed above, N-terminal truncations as short as 36 amino acids(hENG(61-346)-hFc) were found to abolish ligand binding to ENGpolypeptides. To anticipate the effect of even shorter N-terminaltruncations on ligand binding, the secondary structure for the humanendoglin orphan domain was predicted computationally with a modifiedPsipred version 3 (Jones, 1999, J Mol Biol 292:195-202). The analysisindicates that ordered secondary structure within the ENG polypeptideregion defined by amino acids 26-60 of SEQ ID NO: 1 is limited to afour-residue beta strand predicted with high confidence at positions42-45 of SEQ ID NO: 1 and a two-residue beta strand predicted with verylow confidence at positions 28-29 of SEQ ID NO: 1. Accordingly, ENGpolypeptide variants beginning at amino acids 27 or 28 and optionallythose beginning at any of amino acids 29-42 of SEQ ID NO: 1 are likelyto retain important structural elements and ligand binding.

Example 11 Potency of ENG-Fc Variants in a Cell-Based Assay

A reporter-gene assay in A204 cells was used to determine the potencywith which hENG-hFc fusion proteins inhibit signaling by BMP-9 andBMP-10. This assay is based on a human rhabdomyosarcoma cell linetransfected with a pGL3 BRE-luciferase reporter plasmid (Korchynskyi etal, 2002, J Biol Chem 277: 4883-4891), as well as a Renilla reporterplasmid (pRLCMV-luciferase) to control for transfection efficiency. BREmotifs are present in BMP-responsive genes (containing a Id1 promoter),so this vector is of general use for factors signaling through Smad1and/or Smad5. In the absence of ENG-Fc fusion proteins, BMP-9 and BMP-10dose-dependently stimulate signaling in A204 cells.

On the first day of the assay, A204 cells (ATCC® number: HTB-82™;depositor: DJ Giard) were distributed in 48-well plates at 10⁵ cells perwell. On the next day, a solution containing 12 μg pGL3 BRE-luciferase,0.1 μg pRLCMV-luciferase, 30 μl Fugene 6 (Roche Diagnostics), and 970 μlOptiMEM (Invitrogen) was preincubated for 30 min at room temperaturebefore addition to 24 ml of assay buffer (McCoy's medium supplementedwith 0.1% BSA). This mixture was applied to the plated cells (500μl/well) for incubation overnight at 37° C. On the third day, medium wasremoved and replaced with test substances (250 μl/well) diluted in assaybuffer. After an overnight incubation at 37° C., the cells were rinsedand lysed with passive lysis buffer (Promega E1941) and frozen at −70°C. Prior to assay, the plates were warmed to room temperature withgentle shaking. Cell lysates were transferred in duplicate to achemoluminescence plate (96-well) and analyzed in a luminometer withreagents from a Dual-Luciferase Reporter Assay system (Promega E1980) todetermine normalized luciferase activity.

Results indicate that hENG-hFc proteins are potent inhibitors ofcellular signaling mediated by BMP-9 and BMP-10. As shown in the tablebelow, the full-length construct hENG(26-586)-hFc inhibits signaling byBMP-9 and BMP-10 with IC₅₀ values in the sub-nanomolar and low-nanomolarranges, respectively. Moreover, truncated variants hENG(26-359)-hFc andhENG(26-346)-hFc were both more potent than hENG(26-586)-hFc.

IC₅₀ (nM) Construct hBMP-9 hBMP-10 hENG(26-586)-hFc 0.26 7.9hENG(26-359)-hFc 0.16 3.5 hENG(26-346)-hFc 0.19 4.6

Example 12 Truncated Variant hENG(26-359)-hFc Inhibits VEGF-InducibleAngiogenesis in a CAM Assay

Applicants investigated effects of the truncated varianthENG(26-359)-hFc on angiogenesis in the same CAM assay system describedin Example 6, in which VEGF is used to induce angiogenesis. The numberof additional blood vessels induced by VEGF treatment (50 ng daily) wasdecreased by 75% with concurrent hENG(26-359)-hFc (SEQ ID NO: 25; 20 μgdaily) (FIG. 38). SPR-based studies confirmed that VEGF does not bindhENG(26-359)-hFc, and thus effects of this variant on angiogenesis inthe present CAM experiment were not due to a direct interaction betweenthe fusion protein and VEGF. Note that, for hENG(26-359)-hFc, a dose of10 μg corresponds to the dose of 14 μg used for the longer ENG-Fcconstructs tested in Example 6, based on the theoretical molecularweight of each construct. Thus, the truncated variant hENG(26-359)-hFcdisplayed equivalent, if not greater, effectiveness in inhibitingVEGF-inducible angiogenesis compared to ENG constructs with full-lengthECD (FIG. 23) in this same assay system.

Example 13 Truncated Variant hENG(26-346)-hFc Inhibits Angiogenesis in aMouse Angioreactor Assay

Truncated variant hENG(26-346)-hFc was tested in the same mouseangioreactor assay described in Example 7. Angioreactors were implantedsubcutaneously in athymic nude mice (four per mouse), and mice weretreated daily with hENG(26-346)-hFc (10 mg/kg, s.c.) or vehicle(Tris-buffered saline) for 11 days, at which time the mice were injectedwith fluorescein isothiocyanate (FITC)-labeled dextran (20 mg/kg, i.v.)and euthanized 20 min later. The quantity of FITC-dextran contained ineach angioreactor was then measured as an index of blood vesselformation. As shown in FIG. 39, addition of the growth factors (GF)FGF-2 and VEGF to the angioreactors led to a significant increase invascularization, whereas concurrent administration of hENG(26-346)-hFcprevented this increase completely. SPR-based studies confirmed thathENG(26-346)-hFc binds neither FGF-2 nor VEGF, thereby excluding thepossibility that effects of hENG(26-346)-hFc on inducible angiogenesisin the present experiment were due to a direct interaction between thefusion protein and either FGF-2 or VEGF. The present results in thismammalian assay system complement those obtained for the truncatedvariant hENG(26-359)-hFc in a CAM assay (Example 12). Together, theydemonstrate anti-angiogenic activity in vivo of ENG-Fc fusion proteinsincorporating preferred truncations of the ENG extracellular domain.

Example 14 Longer In Vivo Half-Life of Truncated VarianthENG(26-346)-hFc

Applicants conducted a modified pharmacokinetic study to determine thewhole-body elimination half-life of hENG(26-346)-hFc and compared it tothat of the full-length protein mENG(27-581)-mFc. hENG(26-346)-hFcprotein was fluorescently labeled with Alexa Fluor® 750 dye using aSAIVI™ (small animal in vivo imaging) Rapid Antibody Labeling kitaccording to instructions of the manufacturer (Invitrogen™). Labeledprotein was separated from free label by size exclusion chromatography.Athymic nude mice (n=3, 17-20 g) were injected with labeledhENG(26-346)-hFc (2 mg/kg, s.c.), and whole-body imaging was performedwith an IVIS imaging system (Xenogen®/Caliper Life Sciences) todetermine fusion protein levels at 2, 4, 6, 8, 24, 32, 48, and 72 h postinjection. The mean elimination half-life of hENG(26-346)-hFc was 26.5h, which is 20% longer than the 22 h half-life of mENG(27-581)-mFcdetermined in a similar study.

Example 15 Effect of ENG-Fc Proteins on Tumor Growth in Mouse XenograftModels

ENG-Fc proteins were tested in two different mouse xenograft models todetermine whether these proteins can inhibit tumor growth. In the firstexperiment, athymic nude mice were injected subcutaneously at 6 weeks ofage with 10⁶ 4 T1 mammary carcinoma cells (ATCC® number: CRL-2539™;depositor: BA Pulaski). Mice (n=10 per group) were dosed daily (s.c.)with mENG(27-581)-mFc (10 mg/kg) or vehicle (Tris-buffered saline).Tumors were measured manually with digital calipers, and tumor volumewas calculated according to the formula: volume=0.5(length)(width²). Asshown in FIG. 40, treatment with mENG(27-581)-mFc reduced tumor volumeby 45% compared to vehicle by day 24 post implantation.

ENG-Fc fusion proteins were also tested in a Colon-26 carcinomaxenograft model. BALB/c mice were injected subcutaneously at 7 weeks ofage with 1.5×10⁶ Colon-26 carcinoma cells (ATCC® number: CRL-2638™;depositor: N Restifo). Mice (n=10 per group) were dosed daily (s.c.)with mENG(27-581)-mFc (at 1, 10, or 30 mg/kg) or vehicle (Tris-bufferedsaline). Tumor volume was determined as described above. As shown inFIG. 41, mENG(27-581)-mFc treatment caused a dose-dependent reduction intumor volume, with decreases of 55% and nearly 70% compared to vehicleat doses of 10 mg/kg and 30 mg/kg, respectively, by day 58 postimplantation. Thus, mENG(27-581)-mFc markedly slowed the growth of twodifferent tumor types in mouse xenograft models, consistent with theaforementioned antiangiogenic activity of fusion proteins incorporatingthe full-length murine ENG extracellular domain (Examples 5-7). In apreliminary experiment, the truncated variant hENG(26-346) also slowedtumor growth compared to vehicle in the Colon-26 xenograft model,consistent with the antiangiogenic activity of this variant in the mouseangioreactor assay (Example 13).

Taken together, the aforementioned results demonstrate that fusionproteins comprising the full-length ENG ECD, and certain truncatedvariants thereof, display high-affinity binding to BMP-9 and BMP-10 butnot a variety of other TGFI3-family ligands, including TGFI3-1 andTGFI3-3. These ENG polypeptides can inhibit angiogenesis and tumorgrowth in model systems and thus have the potential to treat patientswith unwanted angiogenesis, including those with cancer. Compared toconstructs comprising the full-length ENG ECD, the truncated ENGpolypeptides hENG(26-346)-hFc and/or hENG(26-359)-hFc displayed higherpotency and improved performance on several other key parameters (seesummary table below).

ECD Polypeptide in Fusion Protein (CHO cell derived) Full length ECD -Human 26-586 or Parameter Murine 27-581 Human 26-359 Human 26-346Expression Quantity 31 mg/L 9 mg/L 90 mg/L Quality 96% monomeric 84%monomeric 96% monomeric Binding affinity BMP-9 33 pM 4.2 pM 4.3 pM(K_(D)) BMP-10 490 pM 86 pM 140 pM Dissociation rate BMP-9 25 × 10⁻⁴ s⁻¹3.5 × 10⁻⁴ s⁻¹ 2.4 × 10⁻⁴ s⁻¹ (k_(d)) BMP-10 110 × 10⁻⁴ s⁻¹ 23 × 10⁻⁴s⁻¹  28 × 10⁻⁴ s⁻¹ Potency BMP-9 0.26 nM 0.16 nM 0.19 nM (cell-basedIC₅₀) BMP-10 7.9 nM 3.5 nM 4.6 nM Elimination half-life 22 h — 26.5 hAnti-angiogenesis HUVEC Yes — — activity CAM  65% inhibition 75%inhibition — Angioreactor 100% inhibition — 100% inhibition Anti-tumor4T1 tumor Yes — — activity Colon-26 Yes — Yes tumor Dose-dependent — Notinvestigated

Variant hENG(26-346)-hFc, in particular, possessed a superiorcombination of attributes, with higher potency, stronger bindingaffinity, slower dissociation rate, longer elimination half-life, andbetter protein production than full-length ENG ECD constructs. As ligandtraps, truncated ENG polypeptides should preferably exhibit a slow rateof ligand dissociation, so the ten-fold reduction in the BMP-9dissociation rate for hENG(26-346)-hFc compared to the full-lengthconstruct is highly desirable. The variant hENG(26-378)-hFc displayedBMP-9 binding properties (affinity and dissociation rate) intermediatebetween hENG(26-346)-hFc and hENG(26-359)-hFc, on one hand, andhENG(26-437)-hFc, on the other, with hENG(26-378) more closelyresembling the shorter constructs.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject inventions are explicitlydisclosed herein, the above specification is illustrative and notrestrictive. Many variations of the inventions will become apparent tothose skilled in the art upon review of this specification and theclaims below. The full scope of the inventions should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

1. An endoglin polypeptide comprising an amino acid sequence at least95% identical to amino acids 42-333 of SEQ ID NO: 1, wherein theendoglin polypeptide does not include a sequence consisting of aminoacids 379-430 of SEQ ID NO:
 1. 2. The endoglin polypeptide of claim 1,wherein the endoglin polypeptide comprises an amino acid sequence atleast 95% identical to a sequence beginning at an amino acidcorresponding to any of positions 26-42 of SEQ ID NO: 1 and ending at anamino acid corresponding to any of positions 333-378 of SEQ ID NO:
 1. 3.The endoglin polypeptide of claim 1, comprising an amino acid sequenceat least 95% identical to a sequence selected from a group consistingof: a. amino acids 26-346 of SEQ ID NO: 1, b. amino acids 26-359 of SEQID NO: 1, and c. amino acids 26-378 of SEQ ID NO:
 1. 4. The endoglinpolypeptide of claim 1, wherein the endoglin polypeptide consists of afirst portion consisting of an amino acid sequence at least 95%identical to a sequence selected from a group consisting of: a. aminoacids 26-346 of SEQ ID NO: 1, b. amino acids 26-359 of SEQ ID NO: 1, andc. amino acids 26-378 of SEQ ID NO: 1 and a second portion that isheterologous to SEQ ID NO:1.
 5. The endoglin polypeptide of claim 4,wherein the second portion comprises an Fc portion of an IgG.
 6. Theendoglin polypeptide of claim 1, wherein the endoglin polypeptide doesnot include more than 50 consecutive amino acids from a sequenceconsisting of amino acids 379-586 of SEQ ID NO:
 1. 7. The endoglinpolypeptide of claim 1, wherein the endoglin polypeptide binds humanBMP-9 with an equilibrium dissociation constant (K_(D)) less than 1×10⁻⁹M or a dissociation rate constant (k_(d)) less than 1×10⁻³ s⁻¹.
 8. Theendoglin polypeptide of claim 1, wherein the endoglin polypeptide bindshuman BMP-9 with an equilibrium dissociation constant (K_(D)) less than1×10⁻⁹ M or a dissociation rate constant (k_(d)) less than 5×10⁻⁴ s⁻¹.9. The endoglin polypeptide of claim 1, wherein the endoglin polypeptidebinds human BMP-10 with an equilibrium dissociation constant (K_(D))less than 1×10⁻⁹ M or a dissociation rate constant (k_(d)) less than5×10⁻³ s⁻¹.
 10. (canceled)
 11. An endoglin polypeptide comprising anamino acid sequence encoded by a nucleic acid that hybridizes understringent conditions to a complement of a nucleotide sequence selectedfrom a group consisting of: a. nucleotides 537-1412 of SEQ ID NO: 2[encoding hENG(42-333)], b. nucleotides 121-1035 of SEQ ID NO: 30[encoding hENG(42-346)], c. nucleotides 121-1074 of SEQ ID NO: 26[encoding hENG(42-359)], d. nucleotides 121-1131 of SEQ ID NO: 24[encoding hENG(42-378)], e. nucleotides 73-1035 of SEQ ID NO: 30[encoding hENG(26-346)], f. nucleotides 73-1074 of SEQ ID NO: 26[encoding hENG(26-359)], and g. nucleotides 73-1131 of SEQ ID NO: 24[encoding hENG(26-378)], wherein the endoglin polypeptide does notinclude a sequence consisting of amino acids 379-430 of SEQ ID NO: 1.12. The endoglin polypeptide of claim 1, wherein the endoglinpolypeptide does not bind human TGF-β1, human TGF-β3, human VEGF, orhuman basic fibroblast growth factor (FGF-2).
 13. The endoglinpolypeptide of claim 1, wherein the endoglin polypeptide is a fusionprotein including, in addition to a portion comprising an endoglin aminoacid sequence, one or more polypeptide portions that enhance one or moreof: in vivo stability, in vivo half life, uptake/administration, tissuelocalization or distribution, formation of protein complexes, and/orpurification.
 14. The endoglin polypeptide of claim 13, wherein theendoglin polypeptide includes a portion selected from the groupconsisting of: a constant domain of an immunoglobulin and a serumalbumin.
 15. The endoglin polypeptide of claim 13, wherein the endoglinpolypeptide comprises an immunoglobulin Fc domain.
 16. The endoglinpolypeptide of claim 15, wherein the immunoglobulin Fc domain is joinedto the ENG polypeptide portion by a linker.
 17. The endoglin polypeptideof claim 16, wherein the linker consists of an amino acid sequenceconsisting of SEQ ID NO: 31 (TGGG) or GGG. 18-28. (canceled)
 29. Amethod of inhibiting angiogenesis in a patient in need thereof, themethod comprising administering to the patient an effective amount of anendoglin polypeptide of claim
 1. 30. (canceled)
 31. The method of claim29, wherein the disease is cancer.
 32. The method of claim 31, whereinthe cancer includes types which express undesirably high levels ofBMP-9, BMP-10, or endoglin.
 33. (canceled)
 34. A method of treating aBMP-9 or BMP-10 associated disorder in a patient in need thereof, themethod comprising administering to the patient an effective amount of anendoglin polypeptide of claim 1.